Methods and compositions for increasing efficiency of targeted gene modification using oligonucleotide-mediated gene repair

ABSTRACT

Provided herein include methods and compositions for making targeted changes to a DNA sequence. In various aspects and embodiments, methods and compositions for modifying a DNA sequence in a cell (such as a plant, bacterial, yeast, fungal, algal, or mammalian cell) are provided. In some aspects and embodiments the modification of DNA involves combining gene repair oligonucleotides with approaches that enhance the availability of components of the target cell gene repair mechanisms, such as a DNA cutter.

The present invention is filed under 35 U.S.C. § 371 as the U.S.national phase of International Patent Application No.PCT/US2016/052346, filed Sep. 16, 2016, which designated the UnitedStates and claims benefit of U.S. Provisional Application No.62/293,278, filed Feb. 9, 2016, which is hereby incorporated byreference in its entirety including all tables, figures, and claims andfrom which priority is claimed.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 12, 2019, isnamed CIBUS-29-PCT2US_SeqListing.txt and is 243 kilobytes in size.

FIELD OF THE INVENTION

The instant disclosure relates at least in part to targeted geneticmutations and modifications, including methods and compositions formaking such mutations and modifications.

BACKGROUND

The following discussion is merely provided to aid the reader inunderstanding and is not admitted to describe or constitute prior art tothe present disclosure.

U.S. Pat. No. 6,271,360 discloses methods and compositions for theintroduction of predetermined genetic changes in target genes of aliving cell by introducing an oligodeoxynucleotide encoding thepredetermined change. The oligodeoxynucleotides are effective inmammalian, avian, plant and bacterial cells.

U.S. Pat. No. 8,771,945 discloses vectors and vector systems, some ofwhich encode one or more components of a CRISPR complex, as well asmethods for the design and use of such vectors.

U.S. Pat. No. 8,470,973 “refers to methods for selectively recognizing abase pair in a DNA sequence by a polypeptide, to modified polypeptideswhich specifically recognize one or more base pairs in a DNA sequenceand, to DNA which is modified so that it can be specifically recognizedby a polypeptide and to uses of the polypeptide and DNA in specific DNAtargeting as well as to methods of modulating expression of target genesin a cell.”

SUMMARY

Provided herein include methods and compositions for effecting atargeted genetic change in DNA in a cell. Certain aspects andembodiments relate to improving the efficiency of the targeting ofmodifications to specific locations in genomic or other nucleotidesequences. As described herein, nucleic acids which direct specificchanges to the genome may be combined with various approaches to enhancethe availability of components of the natural repair systems present inthe cells being targeted for modification.

In a first aspect, provided are methods for introducing a gene repairoligonucleobase (GRON)-mediated mutation into a target deoxyribonucleicacid (DNA) sequence in a plant cell. In certain embodiments the methodsmay include, inter alia, culturing the plant cell under conditions thatincrease one or more cellular DNA repair processes prior to, and/orcoincident with, delivery of a GRON into the plant cell; and/or deliveryof a GRON into the plant cell greater than 15 bases in length, the GRONoptionally comprising one or more; or two or more; mutation sites forintroduction into the target DNA.

A “gene repair oligonucleotide” or “GRON” as used herein means anoligonucleobase (e.g., mixed duplex oligonucleotides, non-nucleotidecontaining molecules, single stranded oligodeoxynucleotides, doublestranded oligodeoxynucleotides and other gene repair molecules) that canunder certain conditions direct single, or in some embodiments multiple,nucleotide deletions, insertions or substitutions in a DNA sequence.This oligonucleotide-mediated gene repair editing of the genome maycomprise both non-homology based repair systems (e.g., non-homologousend joining) and homology-based repair systems (e.g., homology-directedrepair). The GRON is typically designed to align in register with agenomic target except for the designed mismatch(es). These mismatchescan be recognized and corrected by harnessing one or more of the cell'sendogenous DNA repair systems. In some embodiments a GRON oroligonucleotide can be designed to contain multiple differences whencompared to the organisms target sequence. These differences may not allaffect the protein sequence translated from said target sequence and inone or more cases be known as silent changes. Numerous variations ofGRON structure, chemistry and function are described elsewhere herein.In various embodiments, a GRON as used herein may have one or moremodifications. For example, a GRON as used herein may have one or moremodifications that attract DNA repair machinery to the targeted(mismatch) site and/or that prevent recombination of part or all of theGRON (other than the desired targeted deletion(s), insertion(s),substitution(s) or the like) into the genomic DNA of the target DNAsequence and/or that increase the stability of the GRON.

In various embodiments, a GRON may have both RNA and DNA nucleotidesand/or other types of nucleobases. In some embodiments, one or more ofthe DNA or RNA nucleotides comprise a modification.

In one aspect, provided is a method of causing a genetic change in aplant cell, wherein the method involves exposing the cell to a DNAcutter and a GRON, for example a GRON that is modified as contemplatedherein. In some embodiments the GRON may be modified such as with a Cy3group, 3PS group, a 2′O-methyl group or other modification such ascontemplated herein. In another aspect, provided is a plant cell thatincludes a DNA cutter and a GRON, for example where the GRON is modifiedsuch as with a Cy3 group, 3PS group, a 2′O-methyl group or othermodification. In some embodiments, the DNA cutter is one or moreselected from a CRISPR, a TALEN, a zinc finger, meganuclease, and aDNA-cutting antibiotic. In some embodiments, the DNA cutter is a CRISPR.In some embodiments, the DNA cutter is a TALEN. In some embodiments, theGRON is between 15 and 60 nucleobases in length; or between 30 and 40nucleobases in length; or between 35 and 45 nucleobases in length; orbetween 20 and 70 nucleobases in length; or between 20 and 200nucleobases in length; or between 30 and 180 nucleobases in length; orbetween 50 and 160 nucleobases in length; or between 70 and 150nucleobases in length; or between 70 and 210 nuceleobases in length; orbetween 80 and 120 nucleobases in length; or between 90 and 110nucleobases in length; or between 95 and 105 nucleobases in length; orbetween 80 and 300 nucleobases in length; or between 90 and 250nucleobases in length; or between 100 and 150 nucleobases in length; orbetween 100 and 200 nucleobases in length; or between 100 and 210nucleobases in length; or between 100 and 300 nucleobases in length; orbetween 150 and 200 nucleobases in length; or between 200 and 300nucleobases in length; or between 250 and 350 nucleobases in length; orbetween 50 and 110 nucleobases in length; or between 50 and 200nucleobases in length; or between 150 and 210 nucleobases in length; orbetween 20 and 1000 nucleobases in length; or between 100 and 1000nucleobases in length; or between 200 and 1000 nucleobases in length; orbetween 300 and 1000 nucleobases in length; or between 400 and 1000nucleobases in length; or between 500 and 1000 nucleobases in length; orbetween 600 and 1000 nucleobases in length; or between 700 and 1000nucleobases in length; or between 800 and 1000 nucleobases in length; orbetween 900 and 1000 nucleobases in length; or between 300 and 800nucleobases in length; or between 400 and 600 nucleobases in length; orbetween 500 and 700 nucleobases in length; or between 600 and 800nucleobases in length; or longer than 30 nucleobases in length; orlonger than 35 nucleobases in length; or longer than 40 nucleobases inlength; or longer than 50 nucleobases in length; or longer than 60nucleobases in length; or longer than 65 nucleobases in length; orlonger than 70 nucleobases in length; or longer than 75 nucleobases inlength; or longer than 80 nucleobases in length; or longer than 85nucleobases in length; or longer than 90 nucleobases in length; orlonger than 95 nucleobases in length; or longer than 100 nucleobases inlength; or longer than 110 nucleobases in length; or longer than 125nucleobases in length; or longer than 150 nucleobases in length; orlonger than 165 nucleobases in length; or longer than 175 nucleobases inlength; or longer than 200 nucleobases in length; or longer than 250nucleobases in length; or longer than 300 nucleobases in length; orlonger than 350 nucleobases in length; or longer than 400 nucleobases inlength; or longer than 450 nucleobases in length; or longer than 500nucleobases in length; or longer than 550 nucleobases in length; orlonger than 600 nucleobases in length; or longer than 700 nucleobases inlength; or longer than 800 nucleobases in length; or longer than 900nucleobases in length.

GRONs may be targeted at both non-coding (NC) and coding (C) regions ofa target gene. By way of example, FIGS. 27 and 28 respectively depictC-GRONs and NC-GRONs suitable for introducing mutations into the ricegenome in order to introduce one or more of the following amino acidsubstitions to the ACCase gene. The convention is to use the amino acidnumbering system for the plastidal ACCase from blackgrass (Alopecurusmyosuroides; Am) as the reference. The ACCase numbering used herein isbased on the numbering for the blackgrass reference sequence ACCaseprotein (SEQ ID NO: 1) or at an analogous amino acid residue in anACCase paralog (V=CY3; H=3′DMT dC CPG). The following table lists ACCasemutations that produce one or more of alloxydim, butroxydim, clethodim,cloproxydim, cycloxydim, sethoxydim, tepraloxydim, tralkoxydim,chlorazifop, clodinafop, clofop, diclofop, fenoxaprop, fenoxaprop-P,fenthiaprop, fluazifop, fluazifop-P, haloxyfop, haloxyfop-P,isoxapyrifop, propaquizafop, quizalofop, quizalofop-P, trifop,pinoxaden, agronomically acceptable salts and esters of any of theseherbicides, and combinations thereof resistant phenotype.

Amino Amino Acid Codon Acid Change Change Change Change Codon I1781AATA > GCT C2088F TGC > TTT ATA > GCC TGC > TTC ATA > GCA C2088G TGC >GGT ATA > GCG TGC > GGC I1781L ATA > CTT TGC > GGA ATA > CTC TGC > GGGATA > CTA C2088H TGC > CAT ATA > CTG TGC > CAC ATA > TTA C2088K TGC >AAA ATA > TTG TGC > AAG I1781M ATA > ATG C2088L TGC > CTT I1781N ATA >AAT TGC > CTC ATA > AAC TGC > CTA I1781S ATA > TCT TGC > CTG ATA > TCCTGC > TTA ATA > TCA TGC > TTG ATA > TCG C2088N TGC > AAT I1781T ATA >ACT TGC > AAC ATA > ACC C2088P TGC > CCT ATA > ACA TGC > CCC ATA > ACGTGC > CCA I1781V ATA > GTT TGC > CCG ATA > GTC C2088Q TGC > CAA ATA >GTA TGC > CAG ATA > GTG C2088R TGC > CGT G1783C GGA > TGT TGC > CGCGGA > TGC TGC > CGA A1786P GCT > CCT TGC > CGG GCT > CCC TGC > AGA GCT >CCA TGC > AGG GCT > CCG C2088S TGC > TCT D2078G GAT > GGT TGC > TCCGAT > GGC TGC > TCA GAT > GGA TGC > TCG GAT > GGG C2088T TGC > ACTD2078K GAT > AAA TGC > ACC GAT > AAG TGC > ACA D2078T GAT > ACT TGC >ACG GAT > ACC C2088V TGC > GTT GAT > ACA TGC > GTC GAT > ACG TGC > GTAS2079F AGC > TTT TGC > GTG AGC > TTC C2088W TGC > TGG K2080E AAG > GAAAAG > GAG

Similarly, FIGS. 29 and 30 respectively depict (coding)C-GRONs and(non-coding)NC-GRONs suitable for introducing mutations into the flaxgenome in order to introduce one or more of the following amino acidsubstitions to the EPSPS gene (with all numbering relative to the aminoacid sequence of the E. coli AroA protein (prokaryotic EPSPS equivalent)(such as those described in U.S. Pat. No. 8,268,622). (V=CY3; H=3′DMT dCCPG). The following table lists EPSPS mutations that produce glyphosateagronomically acceptable salts and esters of any of these herbicides,and combinations thereof resistant phenotype.

Amino Acid Codon Change Change G96A GGA > GCT GGA > GCC GGA > GCA GGA >GCG T971 ACA > ATT ACA > ATC ACA > ATA P101A CCG > GCT CCG > GCC CCG >GCA CCG > GCG P101S CCG > TCT CCG > TCC CCG > TCA CCG > TCG P101T CCG >ACT CCG > ACC CCG > ACA CCG > ACG

The term “CRISPR” as used herein refers to elements; i.e., a cas (CRISPRassociated) gene, transcript (e.g., mRNA) or protein and at least oneCRISPR spacer sequence (Clustered Regularly Interspaced ShortPalindromic Repeats, also known as SPIDRs-SPacer Interspersed DirectRepeats); that when effectively present or expressed in a cell couldeffect cleavage of a target DNA sequence via CRISPR/CAS cellularmachinery such as described in e.g., Cong, L. et al., Science, vol. 339no 6121 pp. 819-823 (2013); Jinek et al, Science, vol. 337:816-821(2013); Wang et al., RNA, vol. 14, pp. 903-913 (2008); Zhang et al.,Plant Physiology, vol. 161, pp. 20-27 (2013), Zhang et al, PCTApplication No. PCT/US2013/074743; and Charpentier et al., PCTApplication No. PCT/US2013/032589. In some embodiments, such as forexample a CRISPR for use in a eukaryotic cell, a CRISPR as contemplatedherein may also include an additional element that includes a sequencefor one or more functional nuclear localization signals. CRISPRs ascontemplated herein can be expressed in, administered to and/or presentin a cell (such as a plant cell) in any of many ways or manifestations.For example a CRISPR as contemplated herein may include or involve oneor more of a CRISPR on a plasmid, a CRISPR nickase on a plasmid, aCRISPRa on a plasmid, or a CRISPRi on a plasmid as follows:

CRISPR on a plasmid: A recombinant expression vector comprising:

(i) a nucleotide sequence encoding a DNA-targeting RNA (e.g., guideRNA), wherein the DNA-targeting RNA comprises:

a, a first segment comprising a nucleotide sequence that iscomplementary to a sequence in a target DNA (e.g., protospacer, spacer,or crRNA); and

b, a second segment that interacts with a site-directed modifyingpolypeptide (e.g., trans-activating crRNA or tracrRNA); and

(ii) a nucleotide sequence encoding the site-directed modifyingpolypeptide (e.g., cas gene), wherein the site-directed polypeptidecomprises:

a, an RNA-binding portion that interacts with the DNA-targeting RNA(e.g., REC lobe); and

b, an activity portion that causes double-stranded breaks within thetarget DNA (e.g., NUC lobe), wherein the site of the double-strandedbreaks within the target DNA is determined by the DNA-targeting RNA.

CRISPR nickase on a plasmid. A recombinant expression vector comprising:

(i) a nucleotide sequence encoding a DNA-targeting RNA (e.g., guideRNA), wherein the DNA-targeting RNA comprises:

a, a first segment comprising a nucleotide sequence that iscomplementary to a sequence in a target DNA (e.g., protospacer, spacer,or crRNA); and

b, a second segment that interacts with a site-directed modifyingpolypeptide (e.g., trans-activating crRNA or tracrRNA); and

(ii) a nucleotide sequence encoding the site-directed modifyingpolypeptide (e.g., cas gene), wherein the site-directed polypeptidecomprises:

a, an RNA-binding portion that interacts with the DNA-targeting RNA(e.g., REC lobe); and

b, an activity portion that causes single-stranded breaks within thetarget DNA (e.g., NUC lobe), wherein the site of the single-strandedbreaks within the target DNA is determined by the DNA-targeting RNA.

CRISPRa on a plasmid. A recombinant expression vector comprising:

(i) a nucleotide sequence encoding a DNA-targeting RNA (e.g., guideRNA), wherein the DNA-targeting RNA comprises:

a, a first segment comprising a nucleotide sequence that iscomplementary to a sequence in a target DNA (e.g., protospacer, spacer,or crRNA); and

b, a second segment that interacts with a site-directed modifyingpolypeptide (e.g., trans-activating crRNA or tracrRNA); and

(ii) a nucleotide sequence encoding the site-directed modifyingpolypeptide (e.g., cas gene), wherein the site-directed polypeptidecomprises:

a, an RNA-binding portion that interacts with the DNA-targeting RNA(e.g., REC lobe); and

b, an activity portion that modulates transcription (e.g., NUC lobe; incertain embodiments increases transcription) within the target DNA,wherein the site of the transcriptional modulation within the target DNAis determined by the DNA-targeting RNA.

CRISPRi on a plasmid. A recombinant expression vector comprising:

(i) a nucleotide sequence encoding a DNA-targeting RNA (e.g., guideRNA), wherein the DNA-targeting RNA comprises:

a, a first segment comprising a nucleotide sequence that iscomplementary to a sequence in a target DNA (e.g., protospacer, spacer,or crRNA); and

b, a second segment that interacts with a site-directed modifyingpolypeptide (e.g., trans-activating crRNA or tracrRNA); and

(ii) a nucleotide sequence encoding the site-directed modifyingpolypeptide (e.g., cas gene), wherein the site-directed polypeptidecomprises:

a, an RNA-binding portion that interacts with the DNA-targeting RNA(e.g., REC lobe); and

b, an activity portion that modulates transcription/translation (e.g.,NUC lobe; in some embodiments decreases transcription/translation)within the target DNA, wherein the site of transcriptional/translationalmodulation within the target DNA is determined by the DNA-targeting RNA.

Each of the CRISPR on a plasmid, CRISPR nickase on a plasmid, CRISPRa ona plasmid, and CRISPRi on a plasmid may in some embodimentsalternatively have one or more appropriate elements be administered,expressed or present in a cell as an RNA (e.g., mRNA) or a proteinrather than on a plasmid. Delivery of protected mRNA may be as describedin Kariko, et al, U.S. Pat. No. 8,278,036.

In some embodiments, each of the CRISPRi and CRISPRa may include adeactivated cas9 (dCas9). A deactivated cas9 still binds to target DNA,but does not have cutting activity. Nuclease-deficient Cas9 can resultfrom D10A and H840A point mutations which inactivates its two catalyticdomains.

In some embodiments, a CRISPRi inhibits transcription initiation orelongation via steric hindrance of RNA Polymerase II. CRISPRi canoptionally be enhanced (CRISPRei) by fusion of a strong repressor domainto the C-terminal end of a dCas9 protein. In some embodiments, arepressor domain recruits and employs chromatin modifiers. In someembodiments, the repressor domain may include, but is not limited todomains as described in Kagale, S. et al., Epigenetics, vol. 6 no 2 pp141-146 (2011):

1. LDLNRPPPVEN (SEQ ID NO: 3) - OsERF3 repressor domain (LxLxPPmotif)(SEQ ID NO: 278) 2. LRLFGVNM (SEQ ID NO: 4) - AtBRD repressordomain (R/KLFGV motif)(SEQ ID NO: 279) 3. LKLFGVWL (SEQ ID NO: 5) -AtHsfB1 repressor domain (R/KLFGV motif)(SEQ ID NO: 279) 4. LDLELRLGFA(SEQ ID NO: 6) - AtSUP repressor domain (EAR motif) 5.ERSNSIELRNSFYGRARTSPWSYGDYDNCQQDHDYLLGFSWPPRSYTCSFCKREFRSAQALGGHMNVHRRDRARLRLQQSPSSSSTPSPPYPNPNYSYSTMANSPPPHHSPLTLFPTLSPPSSPRYRAGLIRSLSPKSKHTPENACKTKKSSLLVEAGEATRFTSKDACKILRNDEIISLELEIGLINESEQDLDLE LRLGFA (SEQ ID NO:7)*- full AtSUP gene containing repressor domain (EAR motif)

In some embodiments, a CRISPRa activation of transcription achieved byuse of dCas9 protein containing a fused C-terminal end transcriptionalactivator. In some embodiments, an activation may include, but is notlimited to VP64 (4×VP16), AtERF98 activation domain, or AtERF98×4concatemers such as described in Cheng, A W et al., Cell Research, pp1-9 (2013); Perez-Pinera, P. et al., Nature Methods, vol. 10 pp 913-976(2013); Maeder, M L. et al., Nature Methods, vol. 10 pp 977-979 (2013)and Mali, P., et al., Nature Biotech., vol. 31 pp 833-838 (2013).

In some embodiments the CRISPR includes a nickase. In certainembodiments, two or more CRISPR nickases are used. In some embodiments,the two or more nickases cut on opposite strands of target nucleic acid.In other embodiments, the two or more nickases cut on the same strand oftarget nucleic acid.

As used herein, “repressor protein” or “repressor” refers to a proteinthat binds to operator of DNA or to RNA to prevent transcription ortranslation, respectively.

As used herein, “repression” refers to inhibition of transcription ortranslation by binding of repressor protein to specific site on DNA ormRNA. In some embodiments, repression includes a significant change intranscription or translation level of at least 1.5 fold, in otherembodiments at least two fold, and in other embodiments at least fivefold.

As used herein, an “activator protein” or “activator” with regard togene transcription and/or translation, refers to a protein that binds tooperator of DNA or to RNA to enhance or increase transcription ortranslation, respectively.

As used herein with regard to gene transcription and/or translation,“activation” with regard to gene transcription and/or translation,refers to enhancing or increasing transcription or translation bybinding of activator protein to specific site on DNA or mRNA. In someembodiments, activation includes a significant change in transcriptionor translation level of at least 1.5 fold, in some embodiments at leasttwo fold, and in some embodiments at least five fold.

In certain embodiments, conditions that increase one or more cellularDNA repair processes may include one or more of: introduction of one ormore sites into the GRON or into the plant cell DNA that are targets forbase excision repair, introduction of one or more sites into the GRON orinto the plant cell DNA that are targets for non-homologous end joining,introduction of one or more sites into the GRON or into the plant cellDNA that are targets for microhomology-mediated end joining,introduction of one or more sites into the GRON or into the plant cellDNA that are targets for homologous recombination, and introduction ofone or more sites into the GRON or into the plant cell DNA that aretargets for effecting repair (e.g., base-excision repair (BER);homologous recombination repair (HR); mismatch repair (MMR);non-homologous end-joining repair (NHEJ) which include classical andalternative NHEJ; and nucleotide excision repair (NER)).

As described herein, GRONs for use herein may include one or more of thefollowing alterations from conventional RNA and DNA nucleotides:

one or more abasic nucleotides;

one or more 8′oxo dA and/or 8′oxo dG nucleotides;

a reverse base at the 3′ end thereof;

one or more 2′O-methyl nucleotides;

one or more RNA nucleotides;

one or more RNA nucleotides at the 5′ end thereof, and in someembodiments 2, 3, 4, 5, 6, 7, 8, 9, 10, or more; wherein one or more ofthe RNA nucleotides may further be modified; one or more RNA nucleotidesat the 3′ end thereof, and in some embodiments 2, 3, 4, 5, 6, 7, 8, 9,10, or more; wherein one or more of the RNA nucleotides may further bemodified;one or more 2′O-methyl RNA nucleotides at the 5′ end thereof, and insome embodiments 2, 3, 4, 5, 6, 7, 8, 9, 10, or more;an intercalating dye;a 5′ terminus cap;a backbone modification selected from the group consisting of aphosphothioate modification, a methyl phosphonate modification, a lockednucleic acid (LNA) modification, a O-(2-methoxyethyl) (MOE)modification, a di PS modification, and a peptide nucleic acid (PNA)modification;one or more intrastrand crosslinks;one or more fluorescent dyes conjugated thereto, and in some embodimentsat the 5′ or 3′ end of the GRON; andone or more bases which increase hybridization energy.This list is not meant to be limiting.

The term “wobble base” as used herein refers to a change in a one ormore nucleotide bases of a reference nucleotide sequence wherein thechange does not change the sequence of the amino acid coded by thenucleotide relative to the reference sequence.

The term “non-nucleotide” or “abasic nucleotide” as use herein refers toany group or compound which can be incorporated into a nucleic acidchain in the place of one or more nucleotide units, including eithersugar and/or phosphate substitutions, and allows the remaining bases toexhibit their enzymatic activity. The group or compound is abasic inthat it does not contain a commonly recognized nucleotide base, such asadenosine, guanine, cytosine, uracil or thymine. It may havesubstitutions for a 2′ or 3′ H or OH as described in the art and herein.

As described herein, in certain embodiments GRON quality and conversionefficiency may be improved by synthesizing all or a portion of the GRONusing nucleotide multimers, such as dimers, trimers, tetramers, etc.improving its purity.

In certain embodiments, the target deoxyribonucleic acid (DNA) sequenceis within a plant cell, for example the target DNA sequence is in theplant cell genome. The plant cell may be non-transgenic or transgenic,and the target DNA sequence may be a transgene or an endogenous gene ofthe plant cell.

In certain embodiments, the conditions that increase one or morecellular DNA repair processes comprise introducing one or more compoundswhich induce single or double DNA strand breaks into the plant cellprior to, or coincident to, or after delivering the GRON into the plantcell. Exemplary compounds are described herein.

The methods and compositions described herein are applicable to plantsgenerally. By way of example only, a plant species may be selected fromthe group consisting of canola, sunflower, corn, tobacco, sugar beet,cotton, maize, wheat (including but not limited to Triticum spp.,Triticum aestivum, Triticum durum Triticum timopheevii, Triticummonococcum, Triticum spelta, Triticum zhukovskyi and Triticum urartu andhybrids thereof), barley (including but not limited to Hordeum vulgareL, Hordeum comosum, Hordeum depressum, Hordeum intercedens, Hordeumjubatum, Hordeum marinum, Hordeum marinum, Hordeum parodii, Hordeumpusillum, Hordeum secalinum, and Hordeum spontaneum), rice (includingbut not limited to Oryza sativa subsp. indica, Oryza sativa subsp.japonica, Oryza sativa subsp. javanica, Oryza sativa subsp. glutinosa(glutinous rice), Oryza sativa Aromatica group (e.g., basmati), andOryza sativa (floating rice group)), alfalfa, barley, sorghum, tomato,mango, peach, apple, pear, strawberry, banana, melon, cassava, potato,carrot, lettuce, onion, soy bean, soya spp, sugar cane, pea, chickpea,field pea, fava bean, lentils, turnip, rutabaga, brussel sprouts, lupin,cauliflower, kale, field beans, poplar, pine, eucalyptus, grape, citrus,triticale, alfalfa, rye (including but not limited to Secale sylvestre,Secale strictum, Secale cereale, Secale vavilovii, Secale africanum,Secale ciliatoglume, Secale ancestrale, and Secale montanum), oats, turf(including but not limited to Turf grass include Zoysia japonica,Agrostris palustris, Poa pratensis, Poa annua, Digitaria sanguinalis,Cyperus rotundus, Kyllinga brevifolia, Cyperus amuricus, Erigeroncanadensis, Hydrocotyle sibthorpioides, Kummerowia striata, Euphorbiahumifusa, and Viola arvensis) and forage grasses, flax, oilseed rape,cotton, mustard, cucumber, morning glory, balsam, pepper, eggplant,marigold, lotus, cabbage, daisy, carnation, tulip, iris, lily,nut-producing plants insofar as they are not already specificallymentioned. These may also apply in whole or in part to all otherbiological systems including but not limited to bacteria, yeast, fungi,algae, and mammalian cells and even their organelles (e.g., mitochondriaand chloroplasts). In some embodiments, the organism or cell is of aspecies selected from the group consisting of Escherichia coli,Mycobacterium smegmatis, Baccillus subtilis, Chlorella, Bacillusthuringiensis, Saccharomyces cerevisiae, Yarrowia lipolytica,Chlamydamonas rhienhardtii, Pichia pastoris, Corynebacterium,Aspergillus niger, and Neurospora crassa. In some embodiments, the yeastis Yarrowia lypolitica. In other embodiments, the yeast is notSaccharomyces cerevisiae. In some embodiments, the plant or plant cellis of a species selected from the group consisting of Arabidopsisthaliana, Solanum tuberosum, Solanum phureja, Oryza sativa, Glycine max,Amaranthus tuberculatus, Linum usitatissimum, and Zea mays. The plantspecies may be selected from the group consisting of monocotyledonousplants of the grass family Poaceae. The family Poaceae may be dividedinto two major clades, the clade containing the subfamiliesBambusoideae, Ehrhartoideae, and Pooideae (the BEP clade) and the cladecontaining the subfamilies Panicoideae, Arundinoideae, Chloridoideae,Centothecoideae, Micrairoideae, Aristidoideae, and Danthonioideae (thePACCMAD clade). The subfamily Bambusoideae includes tribe Oryzeae. Theplant species may relate to plants of the BEP clade, in particularplants of the subfamilies Bambusoideae and Ehrhartoideae. The BET cladeincludes subfamilies Bambusoideae, Ehrhartoideae, and group Triticodaeand no other subfamily Pooideae groups. BET crop plants are plants grownfor food or forage that are members of BET subclade, for example barley,corn, etc.

In certain embodiments, the methods further comprise regenerating aplant having a mutation introduced by the GRON from the plant cell, andmay comprise collecting seeds from the plant.

In related aspects, the present disclosure relates to plant cellscomprising a genomic modification introduced by a GRON according to themethods described herein, a plant comprising a genomic modificationintroduced by a GRON according to the methods described herein, or aseed comprising a genomic modification introduced by a GRON according tothe methods described herein; or progeny of a seed comprising a genomicmodification introduced by a GRON according to the methods describedherein.

Other embodiments of the disclosure will be apparent from the followingdetailed description, exemplary embodiments, and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts BFP to GFP conversion mediated by phosphothioate (PS)labeled GRONs (having 3 PS moieties at each end of the GRON) and5′Cy3/3′idC labeled GRONs. Three independent experiments were done, andin each the PEG treatment was applied in tubes or in a 96-well dish. Fortubes a total of ≈90,000 ppl. in 3 samples of ≈30,000 each per treatmentwere analyzed, for 96-well dishes a total of ≈40,000 ppl. in 4 samplesof ≈10,000 each. Because a non-targeting BFP0/Cy3 GRON was not availableas a control for the targeting GRON BFP-4/NC-Cy3, the Cy3 controlnon-targeting GRON that is used for the conversion of a stop GFP wasused instead (GFP-3/NC-Cy3).

FIG. 2A depicts GRONs (SEQ ID NOS: 38, 339, 33, and 37, respectively, inorder of appearance) comprising RNA/DNA, referred to herein as“2′-O-methyl GRONs.” FIG. 2B depicts GRONs (SEQ ID NOS: 38, 339, 33, and37, respectively, in order of appearance) comprising RNA/DNA, referredto herein as “2′-O-methyl GRONs.”.

FIG. 3 is a schematic of the location on the bfp gene where the BFP5CRISPRs target (SEQ ID NOS: 378 and 379). The GRON used with the BFPCRISPR contains a wobble base shown in italics. This wobble base changesthe nucleotide sequence of the bfp gene, but does not alter the aminoacid sequence. This change in the nucleotide sequence minimizesre-cutting of the bfp gene by the CRISPRs once conversion has happened.The wobble base is located within the PAM sequence itself.

FIG. 4 shows the results of the effect of CRISPRs introduced with eitherthe Cy3 or 3PS GRONs at various lengths, on the percentage of BFP to GFPconversion in a BFP transgenic Arabidopsis thaliana model system. Therewere three replicate for each sample.

FIG. 5 shows the results of the effect of CRISPRs introduced with the3PS GRONs at various lengths, on the percentage of BFP to GFP conversionin a BFP transgenic Arabidopsis thaliana model system. There were threereplicate for each sample.

FIG. 6A discloses GRON “gcugcccgug” (SEQ ID NO: 8) used in Example 9.FIG. 6B discloses GRON “gggcgagggc” (SEQ ID NO: 340) used in Example 9.All GRONs target the bfp gene. The 2′-O-Me GRONs are (A) 71 nt in length(71-mer) or (B) 201 nt in length (201-mer). RNA bases are represented inlower case letters (c,g,a and u) with the letters in bold being baseslabeled with a 2′-O-Me group. There are three different types of 2′-O-MeGRONs: (1) none of the 5′ RNA bases are labeled with a 2′-O-Me group,(2) only the first 5′ RNA base is labeled with a 2′-O-Me group and (3)the first nine RNA bases are labeled with 2′-O-Me groups. 3PS GRONs are(A) 71 nt in length (71-mer) and (B) 201 nt in length (201-mer).

FIG. 7 shows the measurement of mean percentage GFP positive protoplastsfrom an Arabidopsis thaliana BFP transgenic model system as determinedby flow cytometry from 71-mer GRONs. 71-mer GRON concentration is 0.5μM.

FIG. 8 shows the measurement of GFP positive protoplasts fromArabidopsis thaliana BFP transgenic model system as determined by flowcytometry from 201-mer GRONs. 201-mer GRON concentration is 0.16 μM.

FIG. 9 shows the effect of CRISPRs introduced with coding and non-codingGRONs on the mean percentage of GFP positive cells in a BFP transgenicArabidopsis thaliana model system. There were three replicate for eachsample. CR=BFP CRISPR.

FIG. 10 is a schematic of tethering a single stranded GRON or doublestranded DNA to the CRISPR/Cas complex. The DNA, RNA or protein can thenbe tethered to the linker though simple Watson-Crick base pairing. Thismethodology would bring repair molecules in close proximity to nucleaseactivity. An example of the nucleotide sequence of a sgRNA cassette withlinker. This example makes use of a modified single guide RNA (sgRNA)sequence (SEQ ID NOS: 341 and 344) containing a sequence (SEQ ID NO:342) that hybridizes to the target site (SEQ ID NO: 343).

FIG. 11 shows the results of the effect of CRISPRs and GRONs inmediating BFP to GFP conversion in a BFP transgenic Arabidopsis thalianamodel system of spacers of differing lengths. To determine the effect ofCRISPRs and GRONs in mediating BFP to GFP conversion in the BFPtransgenic Arabidopsis thaliana model system at 72h post delivery of DNA(CRISPR plasmids and GRONs). BFP1 spacers of two different lengths 20-ntand 17-nt where directly compared to one another for effectiveness inmediating BFP to GFP conversion. There were three replicates for eachsample. Percentage of GFP positive cells for each treatment wasdetermined using flow cytometry.

FIG. 12 shows the results of the effect of CRISPRs and GRONs inmediating BFP to GFP conversion in a BFP transgenic Arabidopsis thalianamodel system of spacers were encoded on a plasmid (gRNA plasmid) or usedas an amplicon (gRNA amplicon). To determine the effect of CRISPRs andGRONs in mediating BFP to GFP conversion in the BFP transgenicArabidopsis thaliana model system at 72h post delivery of DNA (CRISPRplasmids and GRONs). BFP6 spacers were either encoded on a plasmid (gRNAplasmid) or used as an amplicon (gRNA amplicon) and where directlycompared to one another for effectiveness in mediating BFP to GFPconversion. There were three replicates for each sample. Percentage ofGFP positive cells for each treatment was determined using flowcytometry.

FIG. 13 shows the results of the effect of CRISPRs and GRONs inmediating BFP to GFP conversion in a BFP transgenic Arabidopsis thalianamodel system of unmodified vs. 3PS modified 41-mer GRONs. Measurement ofthe mean percentage of GFP positive protoplasts from Arabidopsisthaliana BFP transgenic model system 72h post delivery of GRONs andCRISPR plasmid was determined by flow cytometry.

FIG. 14 shows the results of next generation sequencing of 3- and 6-weekold Linum usitatissimum (flax) microcalli derived from shoot tipprotoplasts PEG treated with CRISPR plasmid at T=0. 24 hour protoplastsand 3-week old microcalli that are derived from Linum usitatissimumshoot tips were PEG treated with CRISPR plasmid and GRON at T=0.*denotes time after PEG delivery of TALEN plasmids and GRONs. ** denotesprotoplasts and microcalli are not from the same experiment.

FIG. 15 shows the results of next generation sequencing of 3- and 6-weekold Linum usitatissimum microcalli derived from shoot tip protoplastsPEG treated with CRISPR plasmid at T=0.

FIG. 16 a shows the distribution of indels based on size as determinedby deep sequencing in protoplasts treated with CRISPR-Cas plasmid (BC-1)at 72 h post delivery. Indels represented 0.79% of the total reads. FIG.16 b shows BFP to GFP editing measured by the percentage of GFPfluorescing protoplasts identified by flow cytometry 72 h post deliveryof plasmid (BC-1) and GRON (CG-6). Represented data is not normalizedfor transfection efficiency. Error bars are s.e.m. (n=9).

FIG. 17 a shows a comparison of 3PS and unmodified GRONs in BFP to GFPgene editing as measured by flow cytometry at 72 h after delivery ofplasmid (BC-1) and GRONs (CG-1) or (CG-2). FIG. 17 b shows a comparisonof GRON lengths in BFP to GFP gene editing as measured by flow cytometryat 72 hours post delivery of plasmid (BC-2) and GRONs (CG-5) or (CG-8).FIG. 17 c shows a comparison of 3PS to 2′-O-Me GRONs for BFP to GFP geneediting as measured by flow cytometry at 72 h post delivery of plasmid(BC-1) and GRONs (CG-6), (CG-9) or (CG-10). FIG. 17 d shows a comparisonof 3PS to Cy3GRONs in BFP to GFP gene editing as measured by flowcytometry at 72 h post delivery of plasmid (BC-3) and GRONs (CG-3) or(CG-4). Error bars are s.e.m. (n=3). (CG-1): BFP antisense 41 nbunmodified; (CG-2): BFP antisense 41 nb 3PS modified; (CG-3): BFP sense41 nb 3PS modified; (CG-4): BFP sense 41 nb Cy3 modified; (CG-5): BFPsense 60 nb 3PS modified; (CG-6): BFP antisense 201 nb 3PS modified;(CG-8): BFP sense 201 nb 3PS modified; (CG-9): BFP antisense 201 nb2′-O-Me modification on the first 5′ RNA base; (CG-10): BFP antisense201 nb 2′-O-Me modifications on the first nine 5′ RNA bases.

FIG. 18 a shows a distribution of indels based on size as determined bydeep sequencing in Arabidopsis protoplasts treated with TALEN plasmid(BT-1) at 72 h post delivery. Indels represented 0.51% of the totalreads. FIG. 18 b shows BFP to GFP gene editing as measured by flowcytometry at 48 h post delivery of plasmid (BT-1) and GRON (CG-7). FIG.18 c shows a representative distribution of indels based on bp length inL. usitatissimum protoplasts treated with a TALEN (LuET-1) targeting theEPSPS genes 7 d after delivery. Total frequency of indels is 0.50%. FIG.18 d shows L. usitatissimum EPSPS gene editing as measured by deepsequencing at 7 d post delivery of plasmid (LuET-1) and GRON (CG-11)into protoplasts. Percentage of total reads represents the number ofreads containing both T97I and P101A edits as a percentage of the totalreads. Error bars are s.e.m. (n=3). (CG-7): BFP sense 201 nb 3PSmodified; (CG-11): EPSPS sense 144 nb Cy3 modified. [0057] FIG. 19 showseffects of the double strand break inducing antibiotics zeocin andphleomycin on BFP to GFP editing in transgenic A. thaliana protoplasts.Protoplasts were treated with zeocin or phleomycin for 90 min before PEGintroduction of GRON (CG2). Successful editing resulted in GFPfluorescence. Green fluorescing protoplasts were quantified using anAttune Acoustic Focusing Cytometer.

FIG. 20 shows converted BFP transgenic A. thaliana cells five days afterGRON delivery into BFP transgenic protoplasts, targeting the conversionfrom BFP to GFP. Green fluorescence is indicative of BFP-GFP editing. Abrightfield image; B, the same field of view in blue light. Error barsare s.e.m. (n=4); (CG2): BFP antisense 41 nb 3PS modified; (CG12) BFPantisense 41 nb 3PS modified non-targeting. Images were acquired with anImageXpress Micro system (Molecular Devices, Sunnyvale, Calif., USA)Scale bar=20 μm

FIG. 21 a shows a schematic of the CRISPR-Cas plasmid. The mannopinesynthase (Mas) promoter is driving the transcription of the Cas9 genethat is codon optimized for higher plants. The Cas9 gene contains twoSV40 nuclear localization signals (NLS) at either end of the gene and a2X FLAG epitope tag. A. thaliana U6 promoter is driving thetranscription of the gRNA scaffold and transcription is terminated usinga poly(T) signal. FIG. 21 b shows a schematic of the TALEN plasmid. TheMas promoter is driving the transcription of the right and left talearms linked together with a 2A ribosome skipping sequence. A Fok1endonuclease is linked to the 3′ end of each Tale arm. The 5′ end of theleft tale contains a nuclear localization signal (NLS) and a V5 epitopetag. rbcT is the Pisum sativum RBCSE9 gene terminator.

FIG. 22 a shows a BFP gene target region (SEQ ID NO: 345) for theCRISPR-Cas protospacers, BC-1, BC-2 and BC-3 (SEQ ID NOS: 346, 347, and348, respectively) and the TALEN (SEQ ID NO: 349) BT-1, left and righttale arms. The site of BFP to GFP editing is CAC→TAC (H66Y). FIG. 22 bshows an EPSPS gene target region (SEQ ID NO: 350) for the TALEN,LuET-1, left and right tale arms. The site of EPSPS conversions areACA>ATA and CCG>GCG (T97I and P101A).

FIG. 23 shows the amino acid sequence of Alopecurus myosuroides(blackgrass) ACCase gene product (SEQ ID NO: 1).

FIG. 24 shows the amino acid sequence of Escherichia coli EPSPS geneproduct (SEQ ID NO:2).

FIG. 25 shows exemplary analogous EPSPS positions.

FIG. 26A shows an Alopecurus myosuroides plastidal ACCase cDNA sequence(SEQ ID NO: 351). FIG. 26B shows an Alopecurus myosuroides plastidalACCase amino acid sequence (SEQ ID NO: 352). FIG. 26C shows an Oryzasativa plastidal ACCase cDNA sequence (SEQ ID NO: 353). FIG. 26D showsan Oryza sativa plastidal ACCase amino acid sequence (SEQ ID NO: 354).FIG. 26E shows an Oryza sativa plastidal ACCase genomic DNA sequence(SEQ ID NO: 355). FIG. 26F shows an Oryza sativa plastidal ACCaseprotein sequence (SEQ ID NO: 356). FIG. 26G shows an Oryza sativa ACCaseprotein sequence (SEQ ID NO: 357).

FIG. 27(a) shows a diagram of the CRISPR-Cas plasmid, BFP_sgRNA-1. Themannopine synthase (Mas) promoter drives transcription of the plantcodon optimized Cas9 gene that contains two SV40 nuclear localizationsignals (NLS) at the N- and C-terminal and a 3X FLAG epitope tag on theN-terminal. The AtU6-26 promoter drives transcription of the poly (T)terminated gRNA scaffold.

FIG. 27(b) shows diagram depicting the approach used to target locus H66of the BFP transgene (SEQ ID NO: 358). BFP_sgRNA-1. Edited nucleotidechange (C>T) resulting in conversion from BFP fluorescence to GFPfluorescence encoded by BFP/41 GRON (SEQ ID NO: 359) and BFP/101 GRON(SEQ ID NO: 360) result in an edited locus (SEQ ID NO: 360). Othernucleotide changes depicted are silent mutations.

FIG. 27(c) shows a distribution of indels based on size determined bydeep sequencing (n=1).

FIG. 27(d) shows off-target analysis for BFP_sgRNA-1. Indels (SEQ IDNOS: 362 to 366) determined by deep sequencing (n=1), lowercase font aremismatches to BFP_sgRNA-1 target sequence (SEQ ID NO: 361).

FIG. 27(e) shows BFP to GFP editing by BFP_sgRNA-1 and BFP/41 GRON asanalyzed by flow cytometry. R2 gate considered GFP (+) events, errorbars are mean±s.e.m. (n=5).

FIG. 28(a) shows a comparison of 3PS modified (BFP/41/3PS) andunmodified (BFP/41) GRONs in BFP to GFP precision gene editing asanalyzed by flow cytometry. R2 gate considered GFP (+) events, errorbars are mean±s.e.m. (n=5).

FIG. 28(b) shows a comparison of two GRON lengths, 41 nb and 101 nb,with and without 3PS modification in BFP to GFP precision gene editingas analyzed by flow cytometry, error bars are mean±s.e.m. (n=3).

FIG. 29(a) shows a diagram of the CRISPR-Cas9 plasmid EPSPS_sgRNA-2 usedin this study (details are described in FIG. 1 ). Depicted are thetarget locus (SEQ ID NO: 367); sequence encoded by the EPSPS/144 GRON(SEQ ID NO: 368); and the edited locus (SEQ ID NO: 368).

FIG. 29(b) shows Tissue stages in gene editing workflow: 1-flaxprotoplasts, bar 10 μm; 2-microcolony at 3 w, bar 50 μm; 3-micocalli at7 w bar 100 μm; 4-shoot initiation from callus, bar 0.5 c; 5-regeneratedshoots, bar 0.5 cm; 6-regenerated plant in soil.

FIG. 29(c) shows a sequence confirmation of edited EPSPS alleles inregenerated plants Y23 and Z15. Boxed regions show a gene-specificsingle nucleotide polymorphism (SNP), and the ACA>ATA; CCG>GCG edits.Chromatograms are representative of multiple gDNA extractions from eachshoot.

FIG. 30 shows herbicide tolerance to flax calli and regenerated plants.(a) Flax wildtype (wt) callus and callus Y23 with T97I and P101A editsin EPSPS gene 2 were cultured on medium containing glyphosate in a6-well plate. Images were captured 6 days after initiation of treatment.(b) Mean fresh weight of wt and Y23 callus treated with glyphosate after14 days±s.e.m. (n=3). (c) Greenhouse hardened wt and Y23 whole plantswere treated with 10.5 or 21.0 mM glyphosate or surfactant only by sprayapplication. Images were captured 6 days after glyphosate application.This experiment was repeated multiple times with similar results. Bar=2cm

FIG. 31 shows RTDS technology applied to BFP to GFP conversion inArabidopsis. (a) Representation of RTDS technology. (b) Schematicrepresenting BFP to GFP conversion in our BFP transgenic line by RTDS.Converting locus H66 (CAC) to Y66 (TAC) changes the fluorescencecharacter of the transgene from blue to green. Depicted are the targetlocus (SEQ ID NO: 369); sequence encoded by the BFP GRON (SEQ ID NO:370); and the edited locus (SEQ ID NO: 370).

FIG. 32 shows the effect of pre-treatment with phleomycin on RTDSmediated BFP to BFP conversion in Arabidopsis. (a) Protoplasts werepre-treated with 0, 250 or 1000 μg/mL phleomycin for 90 min, and thensubjected to RTDS using either GRON BFP/41 or BFP/41/NT. Error bars aremean±s.e.m. (n=4). (b) Converted A. thaliana cells five days after GRONdelivery. Green fluorescence is indicative of BFP to GFP editing. Abrightfield image; B, the same field of view in blue light. Images wereacquired with an ImageXpress Micro system (Molecular Devices, Sunnyvale,Calif., USA) Scale bar=20 μm

FIG. 33 shows BT-1 TALEN design and target region. (a) BT-1 TALENplasmid. MAS-mannopine synthase promoter; V5-V5 epitope tag; NLS-SV40nuclear localization signal; Fok1 endonuclease is linked to the 3′ endof each Tale arm; rbcT-Pisum sativum RBCSE9 gene terminator. (b)Schematic depicting the BT-1 TALEN target site on the BFP gene. Depictedare the target locus (SEQ ID NO: 371); sequence encoded by the ssODN(SEQ ID NO: 372); and the edited locus (SEQ ID NO: 372). (c) BT-1 TALENdesign and target diagram. The mannopine synthase (MAS) promoter drivesexpression of the TALEN monomers. The rbcT (Pisum sativum) RBCSE9 actsas a gene terminator. A V5 epitope tag and SV40 nuclear localizationsignal (NLS) resides on the N-terminus. The BT-1 Tale left and rightbinding domains are underlined. The site of BFP to GFP conversion is a(C>T) substitution. BT-1 activity produces a DSB that can be repaired byNHEJ or through ssODNs, resulting in deletions and insertions or BFP toGFP precision editing, respectively. Silent substitutions were used todiscourage BT-1 activity after conversion. Depicted are the target locus(SEQ ID NO: 371); sequence encoded by the BFP/41 GRON (SEQ ID NO: 374);sequence encoded by the BFP/1-1 GRON (SEQ ID NO: 375); and the editedlocus (SEQ ID NO: 375).

FIG. 34 shows a CRISPR/Cas9 construct design and target region diagram.A, The mannopine synthase (Mas) promoter drives transcription of theplant codon optimized SpCas9 gene that contains two SV40 nuclearlocalization signals (NLS) at the N- and C-terminal and a 3X FLAGepitope tag on the N-terminal. The rbcT (Pisum sativum) RBCSE9 acts as agene terminator. The AtU6-26 promoter drives transcription of the poly(T) terminated gRNA scaffold. B, Approach used to target the BFPtransgene using BC-1 CRISPR/Cas9. The edited nucleotide change (C>T)results in conversion from BFP to GFP Silent mutations are used to deterBC-1 activity on a converted GFP transgene. Depicted are the targetlocus (SEQ ID NO: 373); sequence encoded by the ssODN (SEQ ID NO: 372);and the edited locus (SEQ ID NO: 372).

FIG. 35 shows BC-1 CRISPR/Cas9 activity in Arabidopsis protoplasts. A,Imprecise NHEJ repairs events in protoplasts treated with BC-1 asdetermined by amplicon deep sequencing (n=1). B, Activity of TALEN BT-1contrasted with CRISPR/Cas9 BC-1 in Arabidopsis protoplasts asdetermined by the percentage of imprecise NHEJ events. C, Off-targetanalysis for BC-1 CRISPR/Cas9. Imprecise NHEJ events at five locihomologous to the BC-1 target sequence were measured by amplicon deepsequencing (n=1). Indels (SEQ ID NOS: 362 to 366) were identified. Basesin lowercase font are mismatches to the BC-1 target sequence (SEQ ID NO:361. D, ssODNs enhance BFP to GFP editing in Arabidopsis protoplaststreated with BC-1. Protoplasts were treated with BFP/41 or BFP/101 withand without CRISPR/Cas9 or BC-1 CRISPR/Cas9 alone. BFP to GFP edits weremeasured by flow cytometry 72h after delivery. Data represent mean±SEM(n=5).

FIG. 36 shows BT-1 TALEN activity in Arabidopsis protoplasts. (a)Distribution of repair events in Arabidopsis protoplasts treated withBT-1 TALEN for 72 h as measured by deep sequencing. (b) Percentage oftotal deletion and insertion repair events with respect to length in bp.(c) Percentage of total deletions≤20 bp by length. (d) Percentage oftotal insertions≤20 bp by length. Error bars are mean±s.e.m. (n=4)

FIG. 37 shows RTDS combined with BT-1 TALEN for editing BFP to GFP inArabidopsis protoplasts. (a) Schematic of the BFP target region for BFPto GFP editing by RTDS. Converting locus H66 (blue font; CAC) to Y66(green font; TAC) changes the fluorescence character of the transgenefrom blue to green. Bases in red are silent mutations used to discourageBT-1 TALEN activity on a corrected GFP gene. (b) BFP to GFP conversionfrequency as determined by cytomety in Arabidopsis protoplasts treatedwith BT-1 TALEN and either BFP/41, BFP/101 or BFP/201 GRON 72 h afterintroduction. GFP gate considered GFP (+) events, error bars aremean±s.e.m. (n=3).

FIG. 38 shows RTDS combined with LuET-1 TALEN for editing the EPSPS lociin flax. (a) LuET-1 TALEN plasmid design. MAS-mannopine synthasepromoter; V5-V5 epitope tag; NLS-SV40 nuclear localization signal; Fok1endonuclease is linked to the 3′ end of each Tale arm; rbcT-Pisumsativum RBCSE9 gene terminator. (b) Schematic depicting the LuET-1 TALENtarget site on conserved sequence of the two flax EPSPS loci. Convertinglocus T97 (ACA) to 197 (ATA) and P101 (CCG) to A101 (GCG) confersglyphosate tolerance. Depicted are the target locus (SEQ ID NO: 376);sequence encoded by the EPSPS/144 GRON (SEQ ID NO: 377); and the editedlocus (SEQ ID NO: 377). (c) Western blot analysis of LuET-1 TALENtransient expression in flax protoplasts 6, 24 and 48h afterintroduction. Reversible stain used as load control. Antibody wasagainst the V5 epitope.

FIG. 39 shows LuET-1 TALEN activity in flax protoplasts. (a)Distribution of repair events in flax microcolonies treated with LuET-1TALEN after 7 d as measured by deep sequencing. (b) Percentage of totaldeletion and insertion repair events with respect to length in bp. (c)Percentage of total deletions≤20 bp by length. (d) Percentage of totalinsertions≤20 bp by length. (n=1).

FIG. 40 shows a diagram depicting TALEN target regions used to generateamplicons for deep sequencing analysis. (a) BT-1 target region on theBFP transgene. BFPF-1 and BFPR-1 primers amplify a region 206 bp inlength that flanks the BT-1 TALEN binding region. (b) LuET-1 targetregion. LuEPF-1 and LuEPR-1 amplify from a conserved region in EPSPSgene 1 and 2, yielding an amplicon flanking the TALEN binding region 194bp in length. SNPs within the amplicon provide gene 1 and 2 specificity.

DETAILED DESCRIPTION

Targeted genetic modification mediated by oligonucleotides is a valuabletechnique for use in the specific alteration of short stretches of DNAto create deletions, short insertions, and point mutations. Thesemethods involve DNA pairing/annealing, followed by a DNArepair/recombination event. First, the nucleic acid anneals with itscomplementary strand in the double-stranded DNA in a process mediated bycellular protein factors. This annealing creates a centrally locatedmismatched base pair (in the case of a point mutation), resulting in astructural perturbation that most likely stimulates the endogenousprotein machinery to initiate the second step in the repair process:site-specific modification of the chromosomal sequence and/or that inorganelles (e.g., mitochondria and chloroplasts). This newly introducedmismatch induces the DNA repair machinery to perform a second repairevent, leading to the final revision of the target site. The presentmethods and compositions in various aspects and embodiments disclosedherein, may improve the methods by providing novel approaches whichincrease the availability of DNA repair components, thus increasing theefficiency and reproducibility of gene repair-mediated modifications totargeted nucleic acids.

Efficient methods for site-directed genomic modifications are desirablefor research, clinical gene therapy, industrial microbiology andagriculture. One approach utilizes triplex-forming oligonucleotides(TFO) which bind as third strands to duplex DNA in a sequence-specificmanner, to mediate directed mutagenesis. Such TFO can act either bydelivering a tethered mutagen, such as psoralen or chlorambucil (Havreet al., Proc Nat'l Acad Sci, U.S.A. 90:7879-7883, 1993; Havre et al., JVirol 67:7323-7331, 1993; Wang et al., Mol Cell Biol 15:1759-1768, 1995;Takasugi et al., Proc Nat'l Acad Sci, U.S.A. 88:5602-5606, 1991;Belousov et al., Nucleic Acids Res 25:3440-3444, 1997), or by bindingwith sufficient affinity to provoke error-prone repair (Wang et al.,Science 271:802-805, 1996).

Another strategy for genomic modification involves the induction ofhomologous recombination between an exogenous DNA fragment and thetargeted gene. This approach has been used successfully to target anddisrupt selected genes in mammalian cells and has enabled the productionof transgenic mice carrying specific gene knockouts (Capeechi et al.,Science 244:1288-1292, 1989; Wagner, U.S. Pat. No. 4,873,191). Thisapproach involves the transfer of selectable markers to allow isolationof the desired recombinants. Without selection, the ratio of homologousto non-homologous integration of transfected DNA in typical genetransfer experiments is low, usually in the range of 1:1000 or less(Sedivy et al., Gene Targeting. W. H. Freeman and Co., New York, 1992).This low efficiency of homologous integration limits the utility of genetransfer for experimental use or gene therapy. The frequency ofhomologous recombination can be enhanced by damage to the target sitefrom UV irradiation and selected carcinogens (Wang et al., Mol Cell Biol8:196-202, 1988) as well as by site-specific endonucleases (Sedivy etal, Gene Targeting, W. H. Freeman and Co., New York, 1992; Rouet et al.,Proc Nat'l Acad Sci, U.S.A. 91:6064-6068, 1994; Segal et al., Proc Nat'lAcad Sci, U.S.A. 92:806-810, 1995). In addition. DNA damage induced bytriplex-directed psoralen photoadducts can stimulate recombinationwithin and between extrachromosomal vectors (Segal et al., Proc Nat'lAcad Sci, U.S.A. 92:806-810, 1995; Faruqi et al., Mol Cell Biol16:6820-6828, 1996; Glazer, U.S. Pat. No. 5,962,426).

Linear donor fragments are more recombinogenic than their circularcounterparts (Folger et al., Mol Cell Biol 2:1372-1387, 1982).Recombination can in certain embodiments also be influenced by thelength of uninterrupted homology between both the donor and targetsites, with short fragments often appearing to be ineffective substratesfor recombination (Rubnitz et al., Mol Cell Biol 4:2253-2258, 1984).Nonetheless, the use of short fragments of DNA or DNA/RNA hybrids forgene correction is the focus of various strategies. (Kunzelmann et al.,Gene Ther 3:859-867, 1996).

“Nucleic acid sequence,” “nucleotide sequence” and “polynucleotidesequence” as used herein refer to an oligonucleotide or polynucleotide,and fragments or portions thereof, and to DNA or RNA of genomic orsynthetic origin which may be single- or double-stranded, and representthe sense or antisense strand.

As used herein, the terms “oligonucleotide” and “oligomer” refer to apolymer of nucleobases of at least about 10 nucleobases and as many asabout 1000 nucleobases.

The terms “DNA-modifying molecule” and “DNA-modifying reagent” as usedherein refer to a molecule which is capable of recognizing andspecifically binding to a nucleic acid sequence in the genome of a cell,and which is capable of modifying a target nucleotide sequence withinthe genome, wherein the recognition and specific binding of theDNA-modifying molecule to the nucleic acid sequence isprotein-independent. The term “protein-independent” as used herein inconnection with a DNA-modifying molecule means that the DNA-modifyingmolecule does not require the presence and/or activity of a proteinand/or enzyme for the recognition of, and/or specific binding to, anucleic acid sequence. DNA-modifying molecules are exemplified, but notlimited to triplex forming oligonucleotides, peptide nucleic acids,polyamides, and oligonucleotides which are intended to promote geneconversion. The DNA-modifying molecules of the present disclosure are incertain embodiments distinguished from the prior art's nucleic acidsequences which are used for homologous recombination (Wong & Capecchi,Molec. Cell. Biol. 7:2294-2295, 1987) in that the prior art's nucleicacid sequences which are used for homologous recombination areprotein-dependent. The term “protein-dependent” as used herein inconnection with a molecule means that the molecule requires the presenceand/or activity of a protein and/or enzyme for the recognition of,and/or specific binding of the molecule to, a nucleic acid sequence.Methods for determining whether a DNA-modifying molecule requires thepresence and/or activity of a protein and/or enzyme for the recognitionof, and/or specific binding to, a nucleic acid sequence are within theskill in the art (see, e.g., Dennis et al. Nucl. Acids Res.27:4734-4742, 1999). For example, the DNA-modifying molecule may beincubated in vitro with the nucleic acid sequence in the absence of anyproteins and/or enzymes. The detection of specific binding between theDNA-modifying molecule and the nucleic acid sequence demonstrates thatthe DNA-modifying molecule is protein-independent. On the other hand,the absence of specific binding between the DNA-modifying molecule andthe nucleic acid sequence demonstrates that the DNA-modifying moleculeis protein-dependent and/or requires additional factors.

“Triplex forming oligonucleotide” (TFO) is defined as a sequence of DNAor RNA that is capable of binding in the major grove of a duplex DNA orRNA helix to form a triple helix. Although the TFO is not limited to anyparticular length, a preferred length of the TFO is 250 nucleotides orless, 200 nucleotides or less, or 100 nucleotides or less, or from 5 to50 nucleotides, or from 10 to 25 nucleotides, or from 15 to 25nucleotides. Although a degree of sequence specificity between the TFOand the duplex DNA is necessary for formation of the triple helix, noparticular degree of specificity is required, as long as the triplehelix is capable of forming. Likewise, no specific degree of avidity oraffinity between the TFO and the duplex helix is required as long as thetriple helix is capable of forming. While not intending to limit thelength of the nucleotide sequence to which the TFO specifically binds inone embodiment, the nucleotide sequence to which the TFO specificallybinds is from 1 to 100, in some embodiments from 5 to 50, yet otherembodiments from 10 to 25, and in other embodiments from 15 to 25,nucleotides. Additionally, “triple helix” is defined as a double-helicalnucleic acid with an oligonucleotide bound to a target sequence withinthe double-helical nucleic acid. The “double-helical” nucleic acid canbe any double-stranded nucleic acid including double-stranded DNA,double-stranded RNA and mixed duplexes of DNA and RNA. Thedouble-stranded nucleic acid is not limited to any particular length.However, in preferred embodiments it has a length of greater than 500bp, in some embodiments greater than 1 kb and in some embodimentsgreater than about 5 kb. In many applications the double-helical nucleicacid is cellular, genomic nucleic acid. The triplex formingoligonucleotide may bind to the target sequence in a parallel oranti-parallel manner.

“Peptide Nucleic Acids,” “polyamides” or “PNA” are nucleic acids whereinthe phosphate backbone is replaced with an N-aminoethylglycine-basedpolyamide structure. PNAs have a higher affinity for complementarynucleic acids than their natural counter parts following theWatson-Crick base-pairing rules. PNAs can form highly stable triplehelix structures with DNA of the following stoichiometry: (PNA)2.DNA.Although the peptide nucleic acids and polyamides are not limited to anyparticular length, a preferred length of the peptide nucleic acids andpolyamides is 200 nucleotides or less, in some embodiments 100nucleotides or less, and in some embodiments from 5 to 50 nucleotideslong. While not intending to limit the length of the nucleotide sequenceto which the peptide nucleic acid and polyamide specifically binds, inone embodiment, the nucleotide sequence to which the peptide nucleicacid and polyamide specifically bind is from 1 to 100, in someembodiments from 5 to 50, yet other embodiments from 5 to 25, and otherembodiments from 5 to 20, nucleotides.

The term “cell” refers to a single cell. The term “cells” refers to apopulation of cells. The population may be a pure population comprisingone cell type. Likewise, the population may comprise more than one celltype. In the present disclosure, there is no limit on the number of celltypes that a cell population may comprise. A cell as used hereinincludes without limitation plant callus cells, cells with and withoutcell walls, prokaryotic cells and eukaryotic cells.

The term “synchronize” or “synchronized,” when referring to a sample ofcells, or “synchronized cells” or “synchronized cell population” refersto a plurality of cells which have been treated to cause the populationof cells to be in the same phase of the cell cycle. It is not necessarythat all of the cells in the sample be synchronized. A small percentageof cells may not be synchronized with the majority of the cells in thesample. A preferred range of cells that are synchronized is between10-100%. A more preferred range is between 30-100%. Also, it is notnecessary that the cells be a pure population of a single cell type.More than one cell type may be contained in the sample. In this regard,only one of cell types may be synchronized or may be in a differentphase of the cell cycle as compared to another cell type in the sample.

The term “synchronized cell” when made in reference to a single cellmeans that the cell has been manipulated such that it is at a cell cyclephase which is different from the cell cycle phase of the cell prior tothe manipulation. Alternatively, a “synchronized cell” refers to a cellthat has been manipulated to alter (i.e., increase or decrease) theduration of the cell cycle phase at which the cell was prior to themanipulation when compared to a control cell (e.g., a cell in theabsence of the manipulation).

The term “cell cycle” refers to the physiological and morphologicalprogression of changes that cells undergo when dividing (i.e.proliferating). The cell cycle is generally recognized to be composed ofphases termed “interphase,” “prophase,” “metaphase,” “anaphase,” and“telophase”. Additionally, parts of the cell cycle may be termed “M(mitosis),” “S (synthesis),” “G0,” “G1 (gap 1)” and “G2 (gap2)”.Furthermore, the cell cycle includes periods of progression that areintermediate to the above named phases.

The term “cell cycle inhibition” refers to the cessation of cell cycleprogression in a cell or population of cells. Cell cycle inhibition isusually induced by exposure of the cells to an agent (chemical,proteinaceous or otherwise) that interferes with aspects of cellphysiology to prevent continuation of the cell cycle.

“Proliferation” or “cell growth” refers to the ability of a parent cellto divide into two daughter cells repeatably thereby resulting in atotal increase of cells in the population. The cell population may be inan organism or in a culture apparatus.

The term “capable of modifying DNA” or “DNA modifying means” refers toprocedures, as well as endogenous or exogenous agents or reagents thathave the ability to induce, or can aid in the induction of, changes tothe nucleotide sequence of a targeted segment of DNA. Such changes maybe made by the deletion, addition or substitution of one or more baseson the targeted DNA segment. It is not necessary that the DNA sequencechanges confer functional changes to any gene encoded by the targetedsequence. Furthermore, it is not necessary that changes to the DNA bemade to any particular portion or percentage of the cells.

The term “nucleotide sequence of interest” refers to any nucleotidesequence, the manipulation of which may be deemed desirable for anyreason, by one of ordinary skill in the art. Such nucleotide sequencesinclude, but are not limited to, coding sequences of structural genes(e.g., reporter genes, selection marker genes, oncogenes, drugresistance genes, growth factors, etc.), and non-coding regulatorysequences that do not encode an mRNA or protein product (e.g., promotersequence, enhancer sequence, polyadenylation sequence, terminationsequence, regulatory RNAs such as miRNA, etc.).

“Amino acid sequence,” “polypeptide sequence,” “peptide sequence” and“peptide” are used interchangeably herein to refer to a sequence ofamino acids.

“Target sequence,” as used herein, refers to a double-helical nucleicacid comprising a sequence greater than 8 nucleotides in length but lessthan 201 nucleotides in length. In some embodiments, the target sequenceis between 8 to 30 bases. The target sequence, in general, is defined bythe nucleotide sequence on one of the strands on the double-helicalnucleic acid.

As used herein, a “purine-rich sequence” or “polypurine sequence” whenmade in reference to a nucleotide sequence on one of the strands of adouble-helical nucleic acid sequence is defined as a contiguous sequenceof nucleotides wherein greater than 50% of the nucleotides of the targetsequence contain a purine base. However, it is preferred that thepurine-rich target sequence contain greater than 60% purine nucleotides,in some embodiments greater than 75% purine nucleotides, in otherembodiments greater than 90% purine nucleotides and yet otherembodiments 100% purine nucleotides.

As used herein, a “pyrimidine-rich sequence” or “polypyrimidinesequence” when made in reference to a nucleotide sequence on one of thestrands of a double-helical nucleic acid sequence is defined as acontiguous sequence of nucleotides wherein greater that 50% of thenucleotides of the target sequence contain a pyrimidine base. However,it is preferred that the pyrimidine-rich target sequence contain greaterthan 60% pyrimidine nucleotides and in some embodiments greater than 75%pyrimidine nucleotides. In some embodiments, the sequence containsgreater than 90% pyrimidine nucleotides and, in other embodiments, is100% pyrimidine nucleotides.

A “variant” of a first nucleotide sequence is defined as a nucleotidesequence which differs from the first nucleotide sequence (e.g., byhaving one or more deletions, insertions, or substitutions that may bedetected using hybridization assays or using DNA sequencing). Includedwithin this definition is the detection of alterations or modificationsto the genomic sequence of the first nucleotide sequence. For example,hybridization assays may be used to detect (1) alterations in thepattern of restriction enzyme fragments capable of hybridizing to thefirst nucleotide sequence when comprised in a genome (i.e., RFLPanalysis), (2) the inability of a selected portion of the firstnucleotide sequence to hybridize to a sample of genomic DNA whichcontains the first nucleotide sequence (e.g., using allele-specificoligonucleotide probes), (3) improper or unexpected hybridization, suchas hybridization to a locus other than the normal chromosomal locus forthe first nucleotide sequence (e.g., using fluorescent in situhybridization (FISH) to metaphase chromosomes spreads, etc.). Oneexample of a variant is a mutated wild type sequence.

The terms “nucleic acid” and “unmodified nucleic acid” as used hereinrefer to any one of the known four deoxyribonucleic acid bases (i.e.,guanine, adenine, cytosine, and thymine). The term “modified nucleicacid” refers to a nucleic acid whose structure is altered relative tothe structure of the unmodified nucleic acid. Illustrative of suchmodifications would be replacement covalent modifications of the bases,such as alkylation of amino and ring nitrogens as well as saturation ofdouble bonds.

As used herein, the terms “mutation” and “modification” and grammaticalequivalents thereof when used in reference to a nucleic acid sequenceare used interchangeably to refer to a deletion, insertion,substitution, strand break, and/or introduction of an adduct. A“deletion” is defined as a change in a nucleic acid sequence in whichone or more nucleotides is absent. An “insertion” or “addition” is thatchange in a nucleic acid sequence which has resulted in the addition ofone or more nucleotides. A “substitution” results from the replacementof one or more nucleotides by a molecule which is a different moleculefrom the replaced one or more nucleotides. For example, a nucleic acidmay be replaced by a different nucleic acid as exemplified byreplacement of a thymine by a cytosine, adenine, guanine, or uridine.Pyrimidine to pyrimidine (e.g. C to T or T to C nucleotidesubstitutions) or purine to purine (e.g. G to A or A to G nucleotidesubstitutions) are termed transitions, whereas pyrimidine to purine orpurine to pyrimidine (e.g. G to T or G to C or A to T or A to C) aretermed transversions. Alternatively, a nucleic acid may be replaced by amodified nucleic acid as exemplified by replacement of a thymine bythymine glycol. Mutations may result in a mismatch. The term “mismatch”refers to a non-covalent interaction between two nucleic acids, eachnucleic acid residing on a different polynucleic acid sequence, whichdoes not follow the base-pairing rules. For example, for the partiallycomplementary sequences 5′-AGT-3′ and 5′-AAT-3′, a G-A mismatch (atransition) is present. The terms “introduction of an adduct” or “adductformation” refer to the covalent or non-covalent linkage of a moleculeto one or more nucleotides in a DNA sequence such that the linkageresults in a reduction (in some embodiments from 10% to 100%, in otherembodiments from 50% to 100%, and in some embodiments from 75% to 100%)in the level of DNA replication and/or transcription.

The term “DNA cutter” refers to a moiety that effects a strand break.Non-limited examples include meganucleases, TALEs/TALENs, antibiotics,zinc fingers and CRISPRs or CRISPR/cas systems.

The term “strand break” when made in reference to a double strandednucleic acid sequence includes a single-strand break and/or adouble-strand break. A single-strand break (a nick) refers to aninterruption in one of the two strands of the double stranded nucleicacid sequence. This is in contrast to a double-strand break which refersto an interruption in both strands of the double stranded nucleic acidsequence, which may result in blunt or staggered ends. Strand breaks maybe introduced into a double stranded nucleic acid sequence eitherdirectly (e.g., by ionizing radiation or treatment with certainchemicals) or indirectly (e.g., by enzymatic incision at a nucleic acidbase).

The terms “mutant cell” and “modified cell” refer to a cell whichcontains at least one modification in the cell's genomic sequence.

The term “portion” when used in reference to a nucleotide sequencerefers to fragments of that nucleotide sequence. The fragments may rangein size from 5 nucleotide residues to the entire nucleotide sequenceminus one nucleic acid residue.

DNA molecules are said to have “5′ ends” and “3′ ends” becausemononucleotides are reacted to make oligonucleotides in a manner suchthat the 5′ phosphate of one mononucleotide pentose ring is attached tothe 3′ oxygen of its neighbor in one direction via a phosphodiesterlinkage. Therefore, an end of an oligonucleotide is referred to as the“5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring. An end of an oligonucleotide is referred toas the “3′ end” if its 3′ oxygen is not linked to a 5′ phosphate ofanother mononucleotide pentose ring. As used herein, a nucleic acidsequence, even if internal to a larger oligonucleotide, also may be saidto have 5′ and 3′ ends. In either a linear or circular DNA molecule,discrete elements are referred to as being “upstream” or 5′ of the“downstream” or 3′ elements. This terminology reflects thattranscription proceeds in a 5′ to 3′ direction along the DNA strand. Thepromoter and enhancer elements which direct transcription of a linkedgene are generally located 5′ or upstream of the coding region. However,enhancer elements can exert their effect even when located 3′ of thepromoter element and the coding region. Transcription termination andpolyadenylation signals are located 3′ or downstream of the codingregion.

The term “recombinant DNA molecule” as used herein refers to a DNAmolecule which is comprised of segments of DNA joined together by meansof molecular biological techniques.

The term “recombinant protein” or “recombinant polypeptide” as usedherein refers to a protein molecule which is expressed using arecombinant DNA molecule.

As used herein, the terms “vector” and “vehicle” are usedinterchangeably in reference to nucleic acid molecules that transfer DNAsegment(s) from one cell to another.

The terms “in operable combination,” “in operable order” and “operablylinked” as used herein refer to the linkage of nucleic acid sequences insuch a manner that a nucleic acid molecule capable of directing thetranscription of a given gene and/or the synthesis of a desired proteinmolecule is produced. The terms also refer to the linkage of amino acidsequences in such a manner so that a functional protein is produced.

The term “transfection” as used herein refers to the introduction offoreign DNA into cells. Transfection may be accomplished by a variety ofmeans known to the art including calcium phosphate-DNA co-precipitation,DEAE-dextran-mediated transfection, polybrene-mediated transfection,electroporation, microinjection, liposome fusion, lipofectin, protoplastfusion, retroviral infection, biolistics (i.e., particle bombardment)and the like.

As used herein, the terms “complementary” or “complementarity” are usedin reference to “polynucleotides” and “oligonucleotides” (which areinterchangeable terms that refer to a sequence of nucleotides) relatedby the base-pairing rules. For example, the sequence “5′-CAGT-3′,” iscomplementary to the sequence “5′-ACTG-3′.” Complementarity can be“partial” or “total”. “Partial” complementarity is where one or morenucleic acid bases is not matched according to the base pairing rules.“Total” or “complete” complementarity between nucleic acids is whereeach and every nucleic acid base is matched with another base under thebase pairing rules. The degree of complementarity between nucleic acidstrands may have significant effects on the efficiency and strength ofhybridization between nucleic acid strands. This may be of particularimportance in amplification reactions, as well as detection methodswhich depend upon binding between nucleic acids. For the sake ofconvenience, the terms “polynucleotides” and “oligonucleotides” includemolecules which include nucleosides.

The terms “homology” and “homologous” as used herein in reference tonucleotide sequences refer to a degree of complementarity with othernucleotide sequences. There may be partial homology or complete homology(i.e., identity). When used in reference to a double-stranded nucleicacid sequence such as a cDNA or genomic clone, the term “substantiallyhomologous” refers to any nucleic acid sequence (e.g., probe) which canhybridize to either or both strands of the double-stranded nucleic acidsequence under conditions of low stringency as described above. Anucleotide sequence which is partially complementary, i.e.,“substantially homologous,” to a nucleic acid sequence is one that atleast partially inhibits a completely complementary sequence fromhybridizing to a target nucleic acid sequence. The inhibition ofhybridization of the completely complementary sequence to the targetsequence may be examined using a hybridization assay (Southern orNorthern blot, solution hybridization and the like) under conditions oflow stringency. A substantially homologous sequence or probe willcompete for and inhibit the binding (i.e., the hybridization) of acompletely homologous sequence to a target sequence under conditions oflow stringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target sequence which lacks even apartial degree of complementarity (e.g., less than about 30% identity);in the absence of non-specific binding the probe will not hybridize tothe second non-complementary target.

Low stringency conditions comprise conditions equivalent to binding orhybridization at 68° C. in a solution consisting of 5×SSPE (43.8 g/lNaCl, 6.9 g/l NaH₂PO₄.H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 withNaOH), 0.1% SDS, 5×Denhardt's reagent (50×Denhardt's contains per 500ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)) and100 gig/ml denatured salmon sperm DNA followed by washing in a solutioncomprising 2.0×SSPE, 0.1% SDS at room temperature when a probe of about100 to about 1000 nucleotides in length is employed.

In addition, conditions which promote hybridization under conditions ofhigh stringency (e.g., increasing the temperature of the hybridizationand/or wash steps, the use of formamide in the hybridization solution,etc.) are well known in the art. High stringency conditions, when usedin reference to nucleic acid hybridization, comprise conditionsequivalent to binding or hybridization at 68° C. in a solutionconsisting of 5×SSPE, 1% SDS, 5×Denhardt's reagent and 100 μg/mldenatured salmon sperm DNA followed by washing in a solution comprising0.1×SSPE and 0.1% SDS at 68° C. when a probe of about 100 to about 1000nucleotides in length is employed.

It is well known in the art that numerous equivalent conditions may beemployed to comprise low stringency conditions; factors such as thelength and nature (DNA, RNA, base composition) of the probe and natureof the target (DNA, RNA, base composition, present in solution orimmobilized, etc.) and the concentration of the salts and othercomponents (e.g., the presence or absence of formamide, dextran sulfate,polyethylene glycol), as well as components of the hybridizationsolution may be varied to generate conditions of low stringencyhybridization different from, but equivalent to, the above listedconditions.

The term “equivalent” when made in reference to a hybridizationcondition as it relates to a hybridization condition of interest meansthat the hybridization condition and the hybridization condition ofinterest result in hybridization of nucleic acid sequences which havethe same range of percent (%) homology. For example, if a hybridizationcondition of interest results in hybridization of a first nucleic acidsequence with other nucleic acid sequences that have from 50% to 70%homology to the first nucleic acid sequence, then another hybridizationcondition is said to be equivalent to the hybridization condition ofinterest if this other hybridization condition also results inhybridization of the first nucleic acid sequence with the other nucleicacid sequences that have from 50% to 70% homology to the first nucleicacid sequence.

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acids using any process by which astrand of nucleic acid joins with a complementary strand through basepairing to form a hybridization complex. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is impacted by such factors as the degree ofcomplementarity between the nucleic acids, stringency of the conditionsinvolved, the Tm of the formed hybrid, and the G:C ratio within thenucleic acids.

As used herein the term “hybridization complex” refers to a complexformed between two nucleic acid sequences by virtue of the formation ofhydrogen bonds between complementary G and C bases and betweencomplementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., Cot or Rotanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized to a solid support (e.g., anylon membrane or a nitrocellulose filter as employed in Southern andNorthern blotting, dot blotting or a glass slide as employed in in situhybridization, including FISH (fluorescent in situ hybridization)).

As used herein, the term “Tm” is used in reference to the “meltingtemperature.” The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. The equation for calculating the Tm ofnucleic acids is well known in the art. As indicated by standardreferences, a simple estimate of the Tm value may be calculated by theequation: Tm=81.5+0.41 (% G+C), when a nucleic acid is in aqueoussolution at 1 M NaCl (see e.g., Anderson and Young, Quantitative FilterHybridization, in Nucleic Acid Hybridization, 1985). Other referencesinclude more sophisticated computations which take structural as well assequence characteristics into account for the calculation of Tm.

As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds such as organic solvents, under which nucleic acidhybridizations are conducted. “Stringency” typically occurs in a rangefrom about Tm−5° C. (5° C. below the melting temperature of the probe)to about 20° C. to 25° C. below Tm. As will be understood by those ofskill in the art, a stringent hybridization can be used to identify ordetect identical polynucleotide sequences or to identify or detectsimilar or related polynucleotide sequences.

The terms “specific binding,” “binding specificity,” and grammaticalequivalents thereof when made in reference to the binding of a firstnucleotide sequence to a second nucleotide sequence, refer to thepreferential interaction between the first nucleotide sequence with thesecond nucleotide sequence as compared to the interaction between thesecond nucleotide sequence with a third nucleotide sequence. Specificbinding is a relative term that does not require absolute specificity ofbinding; in other words, the term “specific binding” does not requirethat the second nucleotide sequence interact with the first nucleotidesequence in the absence of an interaction between the second nucleotidesequence and the third nucleotide sequence. Rather, it is sufficientthat the level of interaction between the first nucleotide sequence andthe second nucleotide sequence is greater than the level of interactionbetween the second nucleotide sequence with the third nucleotidesequence. “Specific binding” of a first nucleotide sequence with asecond nucleotide sequence also means that the interaction between thefirst nucleotide sequence and the second nucleotide sequence isdependent upon the presence of a particular structure on or within thefirst nucleotide sequence; in other words the second nucleotide sequenceis recognizing and binding to a specific structure on or within thefirst nucleotide sequence rather than to nucleic acids or to nucleotidesequences in general. For example, if a second nucleotide sequence isspecific for structure “A” that is on or within a first nucleotidesequence, the presence of a third nucleic acid sequence containingstructure A will reduce the amount of the second nucleotide sequencewhich is bound to the first nucleotide sequence.

As used herein, the term “amplifiable nucleic acid” is used in referenceto nucleic acids which may be amplified by any amplification method. Itis contemplated that “amplifiable nucleic acid” will usually comprise“sample template.”

The terms “heterologous nucleic acid sequence” or “heterologous DNA” areused interchangeably to refer to a nucleotide sequence which is ligatedto a nucleic acid sequence to which it is not ligated in nature, or towhich it is ligated at a different location in nature. Heterologous DNAis not endogenous to the cell into which it is introduced, but has beenobtained from another cell. Generally, although not necessarily, suchheterologous DNA encodes RNA and proteins that are not normally producedby the cell into which it is expressed. Examples of heterologous DNAinclude reporter genes, transcriptional and translational regulatorysequences, selectable marker proteins (e.g., proteins which confer drugresistance), etc.

“Amplification” is defined as the production of additional copies of anucleic acid sequence and is generally carried out using polymerasechain reaction technologies well known in the art (Dieffenbach C W and GS Dveksler (1995) PCR Primer, a Laboratory Manual, Cold Spring HarborPress, Plainview, N.Y.). As used herein, the term “polymerase chainreaction” (“PCR”) refers to the method of K. B. Mullis U.S. Pat. Nos.4,683,195, and 4,683,202, hereby incorporated by reference, whichdescribe a method for increasing the concentration of a segment of atarget sequence in a mixture of genomic DNA without cloning orpurification. The length of the amplified segment of the desired targetsequence is determined by the relative positions of two oligonucleotideprimers with respect to each other, and therefore, this length is acontrollable parameter. By virtue of the repeating aspect of theprocess, the method is referred to as the “polymerase chain reaction”(“PCR”). Because the desired amplified segments of the target sequencebecome the predominant sequences (in terms of concentration) in themixture, they are said to be “PCR amplified.”

With PCR, it is possible to amplify a single copy of a specific targetsequence in genomic DNA to a level detectable by several differentmethodologies (e.g., hybridization with a labeled probe; incorporationof biotinylated primers followed by avidin-enzyme conjugate detection;incorporation of 32P-labeled deoxynucleotide triphosphates, such as dCTPor dATP, into the amplified segment). In addition to genomic DNA, anyoligonucleotide sequence can be amplified with the appropriate set ofprimer molecules. In particular, the amplified segments created by thePCR process itself are, themselves, efficient templates for subsequentPCR amplifications.

One such preferred method, particularly for commercial applications, isbased on the widely used TaqMan® real-time PCR technology, and combinesAllele-Specific PCR with a Blocking reagent (ASB-PCR) to suppressamplification of the wildtype allele. ASB-PCR can be used for detectionof germ line or somatic mutations in either DNA or RNA extracted fromany type of tissue, including formalin-fixed paraffin-embedded tumorspecimens. A set of reagent design rules are developed enablingsensitive and selective detection of single point substitutions,insertions, or deletions against a background of wild-type allele inthousand-fold or greater excess. (Morlan J, Baker J, Sinicropi DMutation Detection by Real-Time PCR: A Simple, Robust and HighlySelective Method. PLoS ONE 4(2): e4584, 2009)

The terms “reverse transcription polymerase chain reaction” and “RT-PCR”refer to a method for reverse transcription of an RNA sequence togenerate a mixture of cDNA sequences, followed by increasing theconcentration of a desired segment of the transcribed cDNA sequences inthe mixture without cloning or purification. Typically, RNA is reversetranscribed using a single primer (e.g., an oligo-dT primer) prior toPCR amplification of the desired segment of the transcribed DNA usingtwo primers.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product which is complementary to a nucleic acid strand isinduced, (i.e., in the presence of nucleotides and of an inducing agentsuch as DNA polymerase and at a suitable temperature and pH). In someembodiments, the primer is single stranded for maximum efficiency inamplification, but may alternatively be double stranded. If doublestranded, the primer is first treated to separate its strands beforebeing used to prepare extension products. In some embodiments, theprimer is an oligodeoxyribonucleotide. The primer must be sufficientlylong to prime the synthesis of extension products in the presence of theinducing agent. The exact lengths of the primers will depend on manyfactors, including temperature, source of primer and the use of themethod.

As used herein, the term “probe” refers to an oligonucleotide (i.e., asequence of nucleotides), whether occurring naturally as in a purifiedrestriction digest or produced synthetically, recombinantly or by PCRamplification, which is capable of hybridizing to anotheroligonucleotide of interest. A probe may be single-stranded ordouble-stranded. Probes are useful in the detection, identification andisolation of particular gene sequences. It is contemplated that anyprobe used in the present disclosure will be labeled with any “reportermolecule,” so that it is detectable in any detection system, including,but not limited to enzyme (e.g., ELISA, as well as enzyme-basedhistochemical assays), fluorescent, radioactive, and luminescentsystems. It is not intended that the present disclosure be limited toany particular detection system or label.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut or nick double-or single-stranded DNA at or near a specific nucleotide sequence, forexample, an endonuclease domain of a type IIS restriction endonuclease(e.g., FokI can be used, as taught by Kim et al., 1996, Proc. Nat'l.Acad. Sci. USA, 6:1 156-60).

As used herein, the term “an oligonucleotide having a nucleotidesequence encoding a gene” means a nucleic acid sequence comprising thecoding region of a gene, i.e. the nucleic acid sequence which encodes agene product. The coding region may be present in either a cDNA, genomicDNA or RNA form. When present in a DNA form, the oligonucleotide may besingle-stranded (i.e., the sense strand) or double-stranded.Additionally “an oligonucleotide having a nucleotide sequence encoding agene” may include suitable control elements such as enhancers,promoters, splice junctions, polyadenylation signals, etc. if needed topermit proper initiation of transcription and/or correct processing ofthe primary RNA transcript. Further still, the coding region of thepresent disclosure may contain endogenous enhancers, splice junctions,intervening sequences, polyadenylation signals, etc.

Transcriptional control signals in eukaryotes comprise “enhancer”elements. Enhancers consist of short arrays of DNA sequences thatinteract specifically with cellular proteins involved in transcription(Maniatis, T. et al., Science 236:1237, 1987). Enhancer elements havebeen isolated from a variety of eukaryotic sources including genes inplant, yeast, insect and mammalian cells and viruses. The selection of aparticular enhancer depends on what cell type is to be used to expressthe protein of interest.

The presence of “splicing signals” on an expression vector often resultsin higher levels of expression of the recombinant transcript. Splicingsignals mediate the removal of introns from the primary RNA transcriptand consist of a splice donor and acceptor site (Sambrook, J. et al.,Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, New York, pp. 16.7-16.8, 1989). A commonly used splicedonor and acceptor site is the splice junction from the 16S RNA of SV40.

Efficient expression of recombinant DNA sequences in eukaryotic cellsrequires expression of signals directing the efficient termination andpolyadenylation of the resulting transcript. Transcription terminationsignals are generally found downstream of the polyadenylation signal andare a few hundred nucleotides in length. The term “poly A site” or “polyA sequence” as used herein denotes a DNA sequence which directs both thetermination and polyadenylation of the nascent RNA transcript. Efficientpolyadenylation of the recombinant transcript is desirable astranscripts lacking a poly A tail are unstable and are rapidly degraded.The poly A signal utilized in an expression vector may be “heterologous”or “endogenous.” An endogenous poly A signal is one that is foundnaturally at the 3′ end of the coding region of a given gene in thegenome. A heterologous poly A signal is one which is isolated from onegene and placed 3′ of another gene.

The term “promoter,” “promoter element” or “promoter sequence” as usedherein, refers to a DNA sequence which when placed at the 5′ end of(i.e., precedes) an oligonucleotide sequence is capable of controllingthe transcription of the oligonucleotide sequence into mRNA. A promoteris typically located 5′ (i.e., upstream) of an oligonucleotide sequencewhose transcription into mRNA it controls, and provides a site forspecific binding by RNA polymerase and for initiation of transcription.

The term “promoter activity” when made in reference to a nucleic acidsequence refers to the ability of the nucleic acid sequence to initiatetranscription of an oligonucleotide sequence into mRNA.

The term “tissue specific” as it applies to a promoter refers to apromoter that is capable of directing selective expression of anoligonucleotide sequence to a specific type of tissue in the relativeabsence of expression of the same oligonucleotide in a different type oftissue. Tissue specificity of a promoter may be evaluated by, forexample, operably linking a reporter gene to the promoter sequence togenerate a reporter construct, introducing the reporter construct intothe genome of a plant or an animal such that the reporter construct isintegrated into every tissue of the resulting transgenic animal, anddetecting the expression of the reporter gene (e.g., detecting mRNA,protein, or the activity of a protein encoded by the reporter gene) indifferent tissues of the transgenic plant or animal. Selectivity neednot be absolute. The detection of a greater level of expression of thereporter gene in one or more tissues relative to the level of expressionof the reporter gene in other tissues shows that the promoter isspecific for the tissues in which greater levels of expression aredetected.

The term “cell type specific” as applied to a promoter refers to apromoter which is capable of directing selective expression of anoligonucleotide sequence in a specific type of cell in the relativeabsence of expression of the same oligonucleotide sequence in adifferent type of cell within the same tissue. The term “cell typespecific” when applied to a promoter also means a promoter capable ofpromoting selective expression of an oligonucleotide in a region withina single tissue. Again, selectivity need not be absolute. Cell typespecificity of a promoter may be assessed using methods well known inthe art, e.g., immunohistochemical staining as described herein.Briefly, tissue sections are embedded in paraffin, and paraffin sectionsare reacted with a primary antibody which is specific for thepolypeptide product encoded by the oligonucleotide sequence whoseexpression is controlled by the promoter. As an alternative to paraffinsectioning, samples may be cryosectioned. For example, sections may befrozen prior to and during sectioning thus avoiding potentialinterference by residual paraffin. A labeled (e.g., peroxidaseconjugated) secondary antibody which is specific for the primaryantibody is allowed to bind to the sectioned tissue and specific bindingdetected (e.g., with avidin/biotin) by microscopy.

The terms “selective expression,” “selectively express” and grammaticalequivalents thereof refer to a comparison of relative levels ofexpression in two or more regions of interest. For example, “selectiveexpression” when used in connection with tissues refers to asubstantially greater level of expression of a gene of interest in aparticular tissue, or to a substantially greater number of cells whichexpress the gene within that tissue, as compared, respectively, to thelevel of expression of, and the number of cells expressing, the samegene in another tissue (i.e., selectivity need not be absolute).Selective expression does not require, although it may include,expression of a gene of interest in a particular tissue and a totalabsence of expression of the same gene in another tissue. Similarly,“selective expression” as used herein in reference to cell types refersto a substantially greater level of expression of, or a substantiallygreater number of cells which express, a gene of interest in aparticular cell type, when compared, respectively, to the expressionlevels of the gene and to the number of cells expressing the gene inanother cell type.

The term “contiguous” when used in reference to two or more nucleotidesequences means the nucleotide sequences are ligated in tandem either inthe absence of intervening sequences, or in the presence of interveningsequences which do not comprise one or more control elements.

As used herein, the terms “nucleic acid molecule encoding,” “nucleotideencoding,” “DNA sequence encoding” and “DNA encoding” refer to the orderor sequence of deoxyribonucleotides along a strand of deoxyribonucleicacid. The order of these deoxyribonucleotides determines the order ofamino acids along the polypeptide (protein) chain. The DNA sequence thuscodes for the amino acid sequence.

The term “isolated” when used in relation to a nucleic acid, as in “anisolated oligonucleotide” refers to a nucleic acid sequence that isseparated from at least one contaminant nucleic acid with which it isordinarily associated in its natural source. Isolated nucleic acid isnucleic acid present in a form or setting that is different from that inwhich it is found in nature. In contrast, non-isolated nucleic acids arenucleic acids such as DNA and RNA which are found in the state theyexist in nature. For example, a given DNA sequence (e.g., a gene) isfound on the host cell chromosome in proximity to neighboring genes; RNAsequences, such as a specific mRNA sequence encoding a specific protein,are found in the cell as a mixture with numerous other mRNAs whichencode a multitude of proteins. However, isolated nucleic acid encodinga polypeptide of interest includes, by way of example, such nucleic acidin cells ordinarily expressing the polypeptide of interest where thenucleic acid is in a chromosomal or extrachromosomal location differentfrom that of natural cells, or is otherwise flanked by a differentnucleic acid sequence than that found in nature. The isolated nucleicacid or oligonucleotide may be present in single-stranded ordouble-stranded form. Isolated nucleic acid can be readily identified(if desired) by a variety of techniques (e.g., hybridization, dotblotting, etc.). When an isolated nucleic acid or oligonucleotide is tobe utilized to express a protein, the oligonucleotide will contain at aminimum the sense or coding strand (i.e., the oligonucleotide may besingle-stranded). Alternatively, it may contain both the sense andanti-sense strands (i.e., the oligonucleotide may be double-stranded).

As used herein, the term “purified” or “to purify” refers to the removalof one or more (undesired) components from a sample. For example, whererecombinant polypeptides are expressed in bacterial host cells, thepolypeptides are purified by the removal of host cell proteins therebyincreasing the percent of recombinant polypeptides in the sample.

As used herein, the term “substantially purified” refers to molecules,either nucleic or amino acid sequences, that are removed from theirnatural environment, isolated or separated, and are at least 60% free,in some embodiments 75% free and other embodiments 90% free from othercomponents with which they are naturally associated. An “isolatedpolynucleotide” is, therefore, a substantially purified polynucleotide.

As used herein the term “coding region” when used in reference to astructural gene refers to the nucleotide sequences which encode theamino acids found in the nascent polypeptide as a result of translationof a mRNA molecule. The coding region is bounded, in eukaryotes, on the5′ side generally by the nucleotide triplet “ATG” which encodes theinitiator methionine and on the 3′ side by one of the three tripletswhich specify stop codons (i.e., TAA, TAG, TGA).

By “coding sequence” is meant a sequence of a nucleic acid or itscomplement, or a part thereof, that can be transcribed and/or translatedto produce the mRNA for and/or the polypeptide or a fragment thereof.Coding sequences include exons in a genomic DNA or immature primary RNAtranscripts, which are joined together by the cell's biochemicalmachinery to provide a mature mRNA. The anti-sense strand is thecomplement of such a nucleic acid, and the encoding sequence can bededuced therefrom.

By “non-coding sequence” is meant a sequence of a nucleic acid or itscomplement, or a part thereof that is not transcribed into amino acid invivo, or where tRNA does not interact to place or attempt to place anamino acid. Non-coding sequences include both intron sequences ingenomic DNA or immature primary RNA transcripts, and gene-associatedsequences such as promoters, enhancers, silencers, etc.

As used herein, the term “structural gene” or “structural nucleotidesequence” refers to a DNA sequence coding for RNA or a protein whichdoes not control the expression of other genes. In contrast, a“regulatory gene” or “regulatory sequence” is a structural gene whichencodes products (e.g., transcription factors) which control theexpression of other genes.

As used herein, the term “regulatory element” refers to a geneticelement which controls some aspect of the expression of nucleic acidsequences. For example, a promoter is a regulatory element whichfacilitates the initiation of transcription of an operably linked codingregion. Other regulatory elements include splicing signals,polyadenylation signals, termination signals, etc.

As used herein, the term “peptide transcription factor binding site” or“transcription factor binding site” refers to a nucleotide sequencewhich binds protein transcription factors and, thereby, controls someaspect of the expression of nucleic acid sequences. For example, Sp-1and API (activator protein 1) binding sites are examples of peptidetranscription factor binding sites.

As used herein, the term “gene” means the deoxyribonucleotide sequencescomprising the coding region of a structural gene. A “gene” may alsoinclude non-translated sequences located adjacent to the coding regionon both the 5′ and 3′ ends such that the gene corresponds to the lengthof the full-length mRNA. The sequences which are located 5′ of thecoding region and which are present on the mRNA are referred to as 5′non-translated sequences. The sequences which are located 3′ ordownstream of the coding region and which are present on the mRNA arereferred to as 3′ non-translated sequences. The term “gene” encompassesboth cDNA and genomic forms of a gene. A genomic form or clone of a genecontains the coding region interrupted with non-coding sequences termed“introns” or “intervening regions” or “intervening sequences.” Intronsare segments of a gene which are transcribed into heterogenous nuclearRNA (hnRNA); introns may contain regulatory elements such as enhancers.Introns are removed or “spliced out” from the nuclear or primarytranscript; introns therefore are absent in the messenger RNA (mRNA)transcript. The mRNA functions during translation to specify thesequence or order of amino acids in a nascent polypeptide. A gene isgenerally a single locus. In a normal diploid organism, a gene has twoalleles. In tetraploid potato, however, each gene has 4 alleles. Insugarcane, which is dodecaploid there can be 12 alleles per gene.Particular examples include flax which has two EPSPS loci each with twoalleles and rice which has a single homomeric plastidal ACCase with twoalleles.

In addition to containing introns, genomic forms of a gene may alsoinclude sequences located on both the 5′ and 3′ end of the sequenceswhich are present on the RNA transcript. These sequences are referred toas “flanking” sequences or regions (these flanking sequences are located5′ or 3′ to the non-translated sequences present on the mRNAtranscript). The 5′ flanking region may contain regulatory sequencessuch as promoters and enhancers which control or influence thetranscription of the gene. The 3′ flanking region may contain sequenceswhich direct the termination of transcription, post-transcriptionalcleavage and polyadenylation.

A “non-human animal” refers to any animal which is not a human andincludes vertebrates such as rodents, non-human primates, ovines,bovines, ruminants, lagomorphs, porcines, caprines, equines, canines,felines, aves, etc. Preferred non-human animals are selected from theorder Rodentia. “Non-human animal” additionally refers to amphibians(e.g. Xenopus), reptiles, insects (e.g. Drosophila) and othernon-mammalian animal species.

As used herein, the term “transgenic” refers to an organism or cell thathas DNA derived from another organism inserted into which becomesintegrated into the genome either of somatic and/or germ line cells ofthe plant or animal. A “transgene” means a DNA sequence which is partlyor entirely heterologous (i.e., not present in nature) to the plant oranimal in which it is found, or which is homologous to an endogenoussequence (i.e., a sequence that is found in the animal in nature) and isinserted into the plant' or animal's genome at a location which differsfrom that of the naturally occurring sequence. Transgenic plants oranimals which include one or more transgenes are within the scope ofthis disclosure. Additionally, a “transgenic” as used herein refers toan organism that has had one or more genes modified and/or “knocked out”(made non-functional or made to function at reduced level, i.e., a“knockout” mutation) by the disclosure's methods, by homologousrecombination, TFO mutation or by similar processes. For example, insome embodiments, a transgenic organism or cell includes inserted DNAthat includes a foreign promoter and/or coding region.

A “transformed cell” is a cell or cell line that has acquired theability to grow in cell culture for multiple generations, the ability togrow in soft agar, and/or the ability to not have cell growth inhibitedby cell-to-cell contact. In this regard, transformation refers to theintroduction of foreign genetic material into a cell or organism.Transformation may be accomplished by any method known which permits thesuccessful introduction of nucleic acids into cells and which results inthe expression of the introduced nucleic acid. “Transformation” includesbut is not limited to such methods as transfection, microinjection,electroporation, nucleofection and lipofection (liposome-mediated genetransfer). Transformation may be accomplished through use of anyexpression vector. For example, the use of baculovirus to introduceforeign nucleic acid into insect cells is contemplated. The term“transformation” also includes methods such as P-element mediatedgermline transformation of whole insects. Additionally, transformationrefers to cells that have been transformed naturally, usually throughgenetic mutation.

As used herein “exogenous” means that the gene encoding the protein isnot normally expressed in the cell. Additionally, “exogenous” refers toa gene transfected into a cell to augment the normal (i.e. natural)level of expression of that gene.

A peptide sequence and nucleotide sequence may be “endogenous” or“heterologous” (i.e., “foreign”). The term “endogenous” refers to asequence which is naturally found in the cell into which it isintroduced so long as it does not contain some modification relative tothe naturally-occurring sequence. The term “heterologous” refers to asequence which is not endogenous to the cell into which it isintroduced. For example, heterologous DNA includes a nucleotide sequencewhich is ligated to, or is manipulated to become ligated to, a nucleicacid sequence to which it is not ligated in nature, or to which it isligated at a different location in nature. Heterologous DNA alsoincludes a nucleotide sequence which is naturally found in the cell intowhich it is introduced and which contains some modification relative tothe naturally-occurring sequence. Generally, although not necessarily,heterologous DNA encodes heterologous RNA and heterologous proteins thatare not normally produced by the cell into which it is introduced.Examples of heterologous DNA include reporter genes, transcriptional andtranslational regulatory sequences, DNA sequences which encodeselectable marker proteins (e.g., proteins which confer drugresistance), etc.

Constructs

The nucleic acid molecules disclosed herein (e.g., site specificnucleases, or guide RNA for CRISPRs) can be used in the production ofrecombinant nucleic acid constructs. In one embodiment, the nucleic acidmolecules of the present disclosure can be used in the preparation ofnucleic acid constructs, for example, expression cassettes forexpression in the plant, microorganism, or animal of interest. Thisexpression may be transient for instance when the construct is notintegrated into the host genome or maintained under the control offeredby the promoter and the position of the construct within the host'sgenome if it becomes integrated.

Expression cassettes may include regulatory sequences operably linked tothe site specific nuclease or guide RNA sequences disclosed herein. Thecassette may additionally contain at least one additional gene to beco-transformed into the organism. Alternatively, the additional gene(s)can be provided on multiple expression cassettes.

The nucleic acid constructs may be provided with a plurality ofrestriction sites for insertion of the site specific nuclease codingsequence to be under the transcriptional regulation of the regulatoryregions. The nucleic acid constructs may additionally contain nucleicacid molecules encoding for selectable marker genes.

Any promoter can be used in the production of the nucleic acidconstructs. The promoter may be native or analogous, or foreign orheterologous, to the plant, microbial, or animal host nucleic acidsequences disclosed herein. Additionally, the promoter may be thenatural sequence or alternatively a synthetic sequence. Where thepromoter is “foreign” or “heterologous” to the plant, microbial, oranimal host, it is intended that the promoter is not found in the nativeplant, microbial, or animal into which the promoter is introduced. Asused herein, a chimeric gene comprises a coding sequence operably linkedto a transcription initiation region that is heterologous to the codingsequence.

The site directed nuclease sequences disclosed herein may be expressedusing heterologous promoters.

Any promoter can be used in the preparation of constructs to control theexpression of the site directed nuclease sequences, such as promotersproviding for constitutive, tissue-preferred, inducible, or otherpromoters for expression in plants, microbes, or animals. Constitutivepromoters include, for example, the core promoter of the Rsyn7 promoterand other constitutive promoters disclosed in WO 99/43 838 and U.S. Pat.No. 6,072,050; the core CaMV 35S promoter (Odell et al. Nature313:810-812; 1985); rice actin (McElroy et al., Plant Cell 2:163-171,1990); ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632, 1989and Christensen et al., Plant Mol. Biol. 18:675-689, 1992); pEMU (Lastet al., Theor. Appl. Genet. 81:581-588, 1991); MAS (Velten et al., EMBOJ. 3:2723-2730, 1984); ALS promoter (U.S. Pat. No. 5,659,026), and thelike. Other constitutive promoters include, for example, U.S. Pat. Nos.5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680;5,268,463; 5,608,142; and 6,177,611.

Tissue-preferred promoters can be utilized to direct site directednuclease expression within a particular plant tissue. Suchtissue-preferred promoters include, but are not limited to,leaf-preferred promoters, root-preferred promoters, seed-preferredpromoters, and stem-preferred promoters. Tissue-preferred promotersinclude Yamamoto et al., Plant J. 12(2):255-265, 1997; Kawamata et al.,Plant Cell Physiol. 38(7):792-803, 1997; Hansen et al., Mol. Gen Genet.254(3):337-343, 1997; Russell et al., Transgenic Res. 6(2):157-168,1997; Rinehart et al., Plant Physiol. 112(3):1331-1341, 1996; Van Campet al., Plant Physiol. 1 12(2):525-535, 1996; Canevascini et al., PlantPhysiol. 112(2): 513-524, 1996; Yamamoto et al., Plant Cell Physiol.35(5):773-778, 1994; Lam, Results Probl. Cell Differ, 20:181-196, 1994;Orozco et al. Plant Mol Biol. 23(6):1129-1138, 1993; Matsuoka et al.,Proc Nat'l. Acad. Sci. USA 90(20):9586-9590, 1993; and Guevara-Garcia etal., Plant J. 4(3):495-505, 1993.

The nucleic acid constructs may also include transcription terminationregions. Where transcription terminations regions are used, anytermination region may be used in the preparation of the nucleic acidconstructs. For example, the termination region may be derived fromanother source (i.e., foreign or heterologous to the promoter). Examplesof termination regions that are available for use in the constructs ofthe present disclosure include those from the Ti-plasmid of A.tumefaciens, such as the octopine synthase and nopaline synthasetermination regions. See also Guerineau et al., Mol. Gen. Genet.262:141-144, 1991; Proudfoot, Cell 64:671-674, 1991; Sanfacon et al.,Genes Dev. 5:141-149, 1991; Mogen et al., Plant Cell 2:1261-1272, 1990;Munroe et al., Gene 91:151-158, 1990; Ballas et al., Nucleic Acids Res.17:7891-7903, 1989; and Joshi et al., Nucleic Acid Res. 15:9627-9639,1987.

In conjunction with any of the aspects, embodiments, methods and/orcompositions disclosed herein, the nucleic acids may be optimized forincreased expression in the transformed plant. That is, the nucleicacids encoding the site directed nuclease proteins can be synthesizedusing plant-preferred codons for improved expression. See, for example,Campbell and Gowri, (Plant Physiol. 92:1-11, 1990) for a discussion ofhost-preferred codon usage. Methods are available in the art forsynthesizing plant-preferred genes. See, for example, U.S. Pat. Nos.5,380,831, and 5,436,391, and Murray et al., Nucleic Acids Res.17:477-498, 1989. See also e.g., Lanza et al., BMC Systems Biology8:33-43, 2014; Burgess-Brown et al., Protein Expr. Purif. 59:94-102,2008; Gustafsson et al., Trends Biotechnol 22:346-353, 2004.

In addition, other sequence modifications can be made to the nucleicacid sequences disclosed herein. For example, additional sequencemodifications are known to enhance gene expression in a cellular host.These include elimination of sequences encoding spurious polyadenylationsignals, exon/intron splice site signals, transposon-like repeats, andother such well-characterized sequences that may be deleterious to geneexpression. The G-C content of the sequence may also be adjusted tolevels average for a target cellular host, as calculated by reference toknown genes expressed in the host cell. In addition, the sequence can bemodified to avoid predicted hairpin secondary mRNA structures.

Other nucleic acid sequences may also be used in the preparation of theconstructs of the present disclosure, for example to enhance theexpression of the site directed nuclease coding sequence. Such nucleicacid sequences include the introns of the maize AdhI, intronl gene(Callis et al., Genes and Development 1:1183-1200, 1987), and leadersequences, (W-sequence) from the Tobacco Mosaic virus (TMV), MaizeChlorotic Mottle Virus and Alfalfa Mosaic Virus (Gallie et al., NucleicAcid Res. 15:8693-8711, 1987; and Skuzeski et al., Plant Mol. Biol.15:65-79, 1990). The first intron from the shrunken-1 locus of maize hasbeen shown to increase expression of genes in chimeric gene constructs.U.S. Pat. Nos. 5,424,412 and 5,593,874 disclose the use of specificintrons in gene expression constructs, and Gallie et al. (Plant Physiol.106:929-939, 1994) also have shown that introns are useful forregulating gene expression on a tissue specific basis. To furtherenhance or to optimize site directed nuclease gene expression, the plantexpression vectors disclosed herein may also contain DNA sequencescontaining matrix attachment regions (MARs). Plant cells transformedwith such modified expression systems, then, may exhibit overexpressionor constitutive expression of a nucleotide sequence of the disclosure.

The expression constructs disclosed herein can also include nucleic acidsequences capable of directing the expression of the site directednuclease sequence to the chloroplast or other organelles and structuresin both prokaryotes and eukaryotes. Such nucleic acid sequences includechloroplast targeting sequences that encodes a chloroplast transitpeptide to direct the gene product of interest to plant cellchloroplasts. Such transit peptides are known in the art. With respectto chloroplast-targeting sequences, “operably linked” means that thenucleic acid sequence encoding a transit peptide (i.e., thechloroplast-targeting sequence) is linked to the site directed nucleasenucleic acid molecules disclosed herein such that the two sequences arecontiguous and in the same reading frame. See, for example, Von Heijneet al., Plant Mol. Biol. Rep. 9:104-126, 1991; Clark et al., J. Biol.Chem. 264:17544-17550, 1989; Della-Cioppa et al., Plant Physiol.84:965-968, 1987; Romer et al., Biochem. Biophys. Res. Commun.196:1414-1421, 1993; and Shah et al., Science 233:478-481, 1986.

Chloroplast targeting sequences are known in the art and include thechloroplast small subunit of ribulose-1,5-bisphosphate carboxylase(Rubisco) (de Castro Silva Filho et al., Plant Mol. Biol. 30:769-780,1996; Schnell et al., J. Biol. Chem. 266(5):3335-3342, 1991);5-(enolpyruvyl)shikimate-3-phosphate synthase (EPSPS) (Archer et al., J.Bioenerg. Biomemb. 22(6):789-810, 1990); tryptophan synthase (Zhao etal., J. Biol. Chem. 270(1 1):6081-6087, 1995); plastocyanin (Lawrence etal., J. Biol. Chem. 272(33):20357-20363, 1997); chorismate synthase(Schmidt et al., J. Biol. Chem. 268(36):27447-27457, 1993); and thelight harvesting chlorophyll a/b binding protein (LHBP) (Lamppa et al.,J. Biol. Chem. 263:14996-14999, 1988). See also Von Heijne et al., PlantMol. Biol. Rep. 9:104-126, 1991; Clark et al., J. Biol. Chem.264:17544-17550, 1989; Della-Cioppa et al., Plant Physiol. 84:965-968,1987; Romer et al., Biochem. Biophys. Res. Commun. 196:1414-1421, 1993;and Shah et al., Science 233: 478-481, 1986.

In conjunction with any of the aspects, embodiments, methods and/orcompositions disclosed herein, the nucleic acid constructs may beprepared to direct the expression of the mutant site directed nucleasecoding sequence from the plant cell chloroplast. Methods fortransformation of chloroplasts are known in the art. See, for example,Svab et al., Proc. Nat'l. Acad. Sci. USA 87:8526-8530, 1990; Svab andMaliga, Proc. Nat'l. Acad. Sci. USA 90:913-917, 1993; Svab and Maliga,EMBO J. 12:601-606, 1993. The method relies on particle gun delivery ofDNA containing a selectable marker and targeting of the DNA to theplastid genome through homologous recombination. Additionally, plastidtransformation can be accomplished by transactivation of a silentplastid-borne transgene by tissue-preferred expression of anuclear-encoded and plastid-directed RNA polymerase. Such a system hasbeen reported in McBride et al. Proc. Nat'l. Acad. Sci. USA91:7301-7305, 1994.

The nucleic acids of interest to be targeted to the chloroplast may beoptimized for expression in the chloroplast to account for differencesin codon usage between the plant nucleus and this organelle. In thismanner, the nucleic acids of interest may be synthesized usingchloroplast-preferred codons. See, for example, U.S. Pat. No. 5,380,831,herein incorporated by reference.

The nucleic acid constructs can be used to transform plant cells andregenerate transgenic plants comprising the site directed nucleasecoding sequences. Numerous plant transformation vectors and methods fortransforming plants are available. See, for example, U.S. Pat. No.6,753,458, An, G. et al., Plant Physiol., 81:301-305, 1986; Fry, J. etal., Plant Cell Rep. 6:321-325, 1987; Block, M., Theor. Appl Genet.76:767-774, 1988; Hinchee et al., Stadler. Genet. Symp. 203212.203-212,1990; Cousins et al., Aust. J. Plant Physiol. 18:481-494, 1991; Chee, P.P. and Slightom, J. L., Gene. 118:255-260, 1992; Christou et al.,Trends. Biotechnol. 10:239-246, 1992; D'Halluin et al., Bio/Technol.10:309-3 14, 1992; Dhir et al., Plant Physiol. 99:81-88, 1992; Casas etal., Proc. Nat'l. Acad Sci. USA 90:11212-11216, 1993; Christou, P., InVitro Cell. Dev. Biol.-Plant 29P:1 19-124, 1993; Davies, et al., PlantCell Rep. 12:180-183, 1993; Dong, J. A. and Mc Hughen, A., Plant Sci.91:139-148, 1993; Franklin, C. I. and Trieu, T. N., Plant. Physiol.102:167, 1993; Golovkin et al., Plant Sci. 90:41-52, 1993; Guo Chin Sci.Bull. 38:2072-2078; Asano, et al., Plant Cell Rep. 13, 1994; Ayeres N.M. and Park, W. D., Crit. Rev. Plant. Sci. 13:219-239, 1994; Barcelo etal., Plant. J. 5:583-592, 1994; Becker, et al., Plant. J. 5:299-307,1994; Borkowska et al., Acta. Physiol Plant. 16:225-230, 1994; Christou,P., Agro. Food. Ind. Hi Tech. 5:17-27, 1994; Eapen et al., Plant CellRep. 13:582-586, 1994; Hartman et al., Bio-Technology 12:919923, 1994;Ritala et al., Plant. Mol. Biol. 24:317-325, 1994; and Wan, Y. C. andLemaux, P. G., Plant Physiol. 104:3748, 1994. The constructs may also betransformed into plant cells using homologous recombination.

The term “wild-type” when made in reference to a peptide sequence andnucleotide sequence refers to a peptide sequence and nucleotide sequence(locus/gene/allele), respectively, which has the characteristics of thatpeptide sequence and nucleotide sequence when isolated from a naturallyoccurring source. A wild-type peptide sequence and nucleotide sequenceis that which is most frequently observed in a population and is thusarbitrarily designated the “normal” or “wild-type” form of the peptidesequence and nucleotide sequence, respectively. “Wild-type” may alsorefer to the sequence at a specific nucleotide position or positions, orthe sequence at a particular codon position or positions, or thesequence at a particular amino acid position or positions.

“Consensus sequence” is defined as a sequence of amino acids ornucleotides that contain identical amino acids or nucleotides orfunctionally equivalent amino acids or nucleotides for at least 25% ofthe sequence. The identical or functionally equivalent amino acids ornucleotides need not be contiguous.

The term “Brassica” as used herein refers to plants of the Brassicagenus. Exemplary Brassica species include, but are not limited to, B.carinata, B. elongate, B. fruticulosa, B. juncea, B. napus, B. narinosa,B. nigra, B. oleracea, B. perviridis, B. rapa (syn B. campestris), B.rupestris, B. septiceps, and B. tournefortii.

A nucleobase is a base, which in certain preferred embodiments is apurine, pyrimidine, or a derivative or analog thereof. Nucleosides arenucleobases that contain a pentosefuranosyl moiety. e.g., an optionallysubstituted riboside or 2′-deoxyriboside. Nucleosides can be linked byone of several linkage moieties, which may or may not containphosphorus. Nucleosides that are linked by unsubstituted phosphodiesterlinkages are termed nucleotides. The term “nucleobase” as used hereinincludes peptide nucleobases, the subunits of peptide nucleic acids, andmorpholine nucleobases as well as nucleosides and nucleotides.

An oligonucleobase is a polymer comprising nucleobases; in someembodiments at least a portion of which can hybridize by Watson-Crickbase pairing to a DNA having the complementary sequence. Anoligonucleobase chain may have a single 5′ and 3′ terminus, which arethe ultimate nucleobases of the polymer. A particular oligonucleobasechain can contain nucleobases of all types. An oligonucleobase compoundis a compound comprising one or more oligonucleobase chains that may becomplementary and hybridized by Watson-Crick base pairing. Ribo-typenucleobases include pentosefuranosyl containing nucleobases wherein the2′ carbon is a methylene substituted with a hydroxyl, alkyloxy orhalogen. Deoxyribo-type nucleobases are nucleobases other than ribo-typenucleobases and include all nucleobases that do not contain apentosefuranosyl moiety.

In certain embodiments, an oligonucleobase strand may include botholigonucleobase chains and segments or regions of oligonucleobasechains. An oligonucleobase strand may have a 3′ end and a 5′ end, andwhen an oligonucleobase strand is coextensive with a chain, the 3′ and5′ ends of the strand are also 3′ and 5′ termini of the chain.

As used herein the term “codon” refers to a sequence of three adjacentnucleotides (either RNA or DNA) constituting the genetic code thatdetermines the insertion of a specific amino acid in a polypeptide chainduring protein synthesis or the signal to stop protein synthesis. Theterm “codon” is also used to refer to the corresponding (andcomplementary) sequences of three nucleotides in the messenger RNA intowhich the original DNA is transcribed.

As used herein, the term “homology” refers to sequence similarity amongproteins and DNA. The term “homology” or “homologous” refers to a degreeof identity. There may be partial homology or complete homology. Apartially homologous sequence is one that has less than 100% sequenceidentity when compared to another sequence.

“Heterozygous” refers to having different alleles at one or more geneticloci in homologous chromosome segments. As used herein “heterozygous”may also refer to a sample, a cell, a cell population or an organism inwhich different alleles at one or more genetic loci may be detected.Heterozygous samples may also be determined via methods known in the artsuch as, for example, nucleic acid sequencing. For example, if asequencing electropherogram shows two peaks at a single locus and bothpeaks are roughly the same size, the sample may be characterized asheterozygous. Or, if one peak is smaller than another, but is at leastabout 25% the size of the larger peak, the sample may be characterizedas heterozygous. In some embodiments, the smaller peak is at least about15% of the larger peak. In other embodiments, the smaller peak is atleast about 10% of the larger peak. In other embodiments, the smallerpeak is at least about 5% of the larger peak. In other embodiments, aminimal amount of the smaller peak is detected.

As used herein, “homozygous” refers to having identical alleles at oneor more genetic loci in homologous chromosome segments. “Homozygous” mayalso refer to a sample, a cell, a cell population or an organism inwhich the same alleles at one or more genetic loci may be detected.Homozygous samples may be determined via methods known in the art, suchas, for example, nucleic acid sequencing. For example, if a sequencingelectropherogram shows a single peak at a particular locus, the samplemay be termed “homozygous” with respect to that locus.

The term “hemizygous” refers to a gene or gene segment being presentonly once in the genotype of a cell or an organism because the secondallele is deleted, or is not present on the homologous chromosomesegment. As used herein “hemizygous” may also refer to a sample, a cell,a cell population or an organism in which an allele at one or moregenetic loci may be detected only once in the genotype.

The term “zygosity status” as used herein refers to a sample, a cellpopulation, or an organism as appearing heterozygous, homozygous, orhemizygous as determined by testing methods known in the art anddescribed herein. The term “zygosity status of a nucleic acid” meansdetermining whether the source of nucleic acid appears heterozygous,homozygous, or hemizygous. The “zygosity status” may refer todifferences in at a single nucleotide position in a sequence. In somemethods, the zygosity status of a sample with respect to a singlemutation may be categorized as homozygous wild-type, heterozygous (i.e.,one wild-type allele and one mutant allele), homozygous mutant, orhemizygous (i.e., a single copy of either the wild-type or mutantallele).

As used herein, the term “RTDS” refers to The Rapid Trait DevelopmentSystem™ (RTDS) developed by Cibus. RTDS is a site-specific genemodification system that is effective at making precise changes in agene sequence without the incorporation of foreign genes or controlsequences.

The term “about” as used herein means in quantitative terms plus orminus 10%. For example, “about 3%” would encompass 2.7-3.3% and “about10%” would encompass 9-11%. Moreover, where “about” is used herein inconjunction with a quantitative term it is understood that in additionto the value plus or minus 10%, the exact value of the quantitative termis also contemplated and described. For example, the term “about 3%”expressly contemplates, describes and includes exactly 3%.

RTDS and Repair Oligonucleotides (GRONs)

This disclosure generally relates to novel methods to improve theefficiency of the targeting of modifications to specific locations ingenomic or other nucleotide sequences. Additionally, this disclosurerelates to target DNA that has been modified, mutated or marked by theapproaches disclosed herein. The disclosure also relates to cells,tissue, and organisms which have been modified by the disclosure'smethods. The present disclosure builds on the development ofcompositions and methods related in part to the successful conversionsystem, the Rapid Trait Development System (RTDS™, Cibus US LLC).

RTDS is based on altering a targeted gene by utilizing the cell's owngene repair system to specifically modify the gene sequence in situ andnot insert foreign DNA and gene expression control sequences. Thisprocedure effects a precise change in the genetic sequence while therest of the genome is left unaltered. In contrast to conventionaltransgenic GMOs, there is no integration of foreign genetic material,nor is any foreign genetic material left in the plant. The changes inthe genetic sequence introduced by RTDS are not randomly inserted. Sinceaffected genes remain in their native location, no random, uncontrolledor adverse pattern of expression occurs.

The RTDS that effects this change is a chemically synthesizedoligonucleotide (GRON) as described herein which may be composed of bothDNA and modified RNA bases as well as other chemical moieties, and isdesigned to hybridize at the targeted gene location to create amismatched base-pair(s). This mismatched base-pair acts as a signal toattract the cell's own natural gene repair system to that site andcorrect (replace, insert or delete) the designated nucleotide(s) withinthe gene. Once the correction process is complete the RTDS molecule isdegraded and the now-modified or repaired gene is expressed under thatgene's normal endogenous control mechanisms.

The methods and compositions disclosed herein can be practiced or madewith “gene repair oligonucleobases” (GRON) having the conformations andchemistries as described in detail herein and below. The “gene repairoligonucleobases” as contemplated herein have also been described inpublished scientific and patent literature using other names including“recombinagenic oligonucleobases;” “RNA/DNA chimeric oligonucleotides;”“chimeric oligonucleotides;” “mixed duplex oligonucleotides” (MDONs);“RNA DNA oligonucleotides (RDOs);” “gene targeting oligonucleotides;”“genoplasts;” “single stranded modified oligonucleotides;” “Singlestranded oligodeoxynucleotide mutational vectors” (SSOMVs); “duplexmutational vectors;” and “heteroduplex mutational vectors.” The generepair oligonucleobase can be introduced into a plant cell using anymethod commonly used in the art, including but not limited to,microcarriers (biolistic delivery), microfibers, polyethylene glycol(PEG)-mediated uptake, electroporation, and microinjection.

In one embodiment, the gene repair oligonucleobase is a mixed duplexoligonucleotides (MDON) in which the RNA-type nucleotides of the mixedduplex oligonucleotide are made RNase resistant by replacing the2′-hydroxyl with a fluoro, chloro or bromo functionality or by placing asubstituent on the 2′-O. Suitable substituents include the substituentstaught by the Kmiec II. Alternative substituents include thesubstituents taught by U.S. Pat. No. 5,334,711 (Sproat) and thesubstituents taught by patent publications EP 629 387 and EP 679 657(collectively, the Martin Applications), which are hereby incorporatedby reference. As used herein, a 2′-fluoro, chloro or bromo derivative ofa ribonucleotide or a ribonucleotide having a T-OH substituted with asubstituent described in the Martin Applications or Sproat is termed a“T-Substituted Ribonucleotide.” As used herein the term “RNA-typenucleotide” means a T-hydroxyl or 2′-Substituted Nucleotide that islinked to other nucleotides of a mixed duplex oligonucleotide by anunsubstituted phosphodiester linkage or any of the non-natural linkagestaught by Kmiec I or Kmiec II. As used herein the term “deoxyribo-typenucleotide” means a nucleotide having a T-H, which can be linked toother nucleotides of a gene repair oligonucleobase by an unsubstitutedphosphodiester linkage or any of the non-natural linkages taught byKmiec I or Kmiec II.

In a particular embodiment of the present disclosure, the gene repairoligonucleobase is a mixed duplex oligonucleotide (MDON) that is linkedsolely by unsubstituted phosphodiester bonds. In alternativeembodiments, the linkage is by substituted phosphodiesters,phosphodiester derivatives and non-phosphorus-based linkages as taughtby Kmiec II. In yet another embodiment, each RNA-type nucleotide in themixed duplex oligonucleotide is a 2′-Substituted Nucleotide. Particularpreferred embodiments of 2′-Substituted Ribonucleotides are 2′-fluoro.T-methoxy, 2′-propyloxy, 2′-allyloxy, 2′-hydroxylethyloxy,2′-methoxyethyloxy, T-fluoropropyloxy and 2′-trifluoropropyloxysubstituted ribonucleotides. More preferred embodiments of2′-Substituted Ribonucleotides are 2′-fluoro, 2′-methoxy,2′-methoxyethyloxy, and 2′-allyloxy substituted nucleotides. In anotherembodiment the mixed duplex oligonucleotide is linked by unsubstitutedphosphodiester bonds.

Although mixed duplex oligonucleotides (MDONs) having only a single typeof 2′-substituted RNA-type nucleotide are more conveniently synthesized,the methods of the disclosure can be practiced with mixed duplexoligonucleotides having two or more types of RNA-type nucleotides. Thefunction of an RNA segment may not be affected by an interruption causedby the introduction of a deoxynucleotide between two RNA-typetrinucleotides, accordingly, the term RNA segment encompasses terms suchas “interrupted RNA segment.” An uninterrupted RNA segment is termed acontiguous RNA segment. In an alternative embodiment an RNA segment cancontain alternating RNase-resistant and unsubstituted 2′-OH nucleotides.The mixed duplex oligonucleotides in some embodiments have fewer than100 nucleotides and other embodiments fewer than 85 nucleotides, butmore than 50 nucleotides. The first and second strands are Watson-Crickbase paired. In one embodiment the strands of the mixed duplexoligonucleotide are covalently bonded by a linker, such as a singlestranded hexa, penta or tetranucleotide so that the first and secondstrands are segments of a single oligonucleotide chain having a single3′ and a single 5′ end. The 3′ and 5′ ends can be protected by theaddition of a “hairpin cap” whereby the 3′ and 5′ terminal nucleotidesare Watson-Crick paired to adjacent nucleotides. A second hairpin capcan, additionally, be placed at the junction between the first andsecond strands distant from the 3′ and 5′ ends, so that the Watson-Crickpairing between the first and second strands is stabilized.

The first and second strands contain two regions that are homologouswith two fragments of the target gene/allele, i.e., have the samesequence as the target gene/allele. A homologous region contains thenucleotides of an RNA segment and may contain one or more DNA-typenucleotides of connecting DNA segment and may also contain DNA-typenucleotides that are not within the intervening DNA segment. The tworegions of homology are separated by, and each is adjacent to, a regionhaving a sequence that differs from the sequence of the target gene,termed a “heterologous region.” The heterologous region can contain one,two or three mismatched nucleotides. The mismatched nucleotides can becontiguous or alternatively can be separated by one or two nucleotidesthat are homologous with the target gene/allele. Alternatively, theheterologous region can also contain an insertion or one, two, three orof five or fewer nucleotides. Alternatively, the sequence of the mixedduplex oligonucleotide may differ from the sequence of the targetgene/allele only by the deletion of one, two, three, or five or fewernucleotides from the mixed duplex oligonucleotide. The length andposition of the heterologous region is, in this case, deemed to be thelength of the deletion, even though no nucleotides of the mixed duplexoligonucleotide are within the heterologous region. The distance betweenthe fragments of the target gene that are complementary to the twohomologous regions is identical to the length of the heterologous regionwhere a substitution or substitutions is intended. When the heterologousregion contains an insertion, the homologous regions are therebyseparated in the mixed duplex oligonucleotide farther than theircomplementary homologous fragments are in the gene/allele, and theconverse is applicable when the heterologous region encodes a deletion.

The RNA segments of the mixed duplex oligonucleotides are each a part ofa homologous region, i.e., a region that is identical in sequence to afragment of the target gene, which segments together in some embodimentscontain at least 13 RNA-type nucleotides and in some embodiments from 16to 25 RNA-type nucleotides or yet other embodiments 18-22 RNA-typenucleotides or in some embodiments 20 nucleotides. In one embodiment,RNA segments of the homology regions are separated by and adjacent to,i.e., “connected by” an intervening DNA segment. In one embodiment, eachnucleotide of the heterologous region is a nucleotide of the interveningDNA segment. An intervening DNA segment that contains the heterologousregion of a mixed duplex oligonucleotide is termed a “mutator segment.”

In another embodiment of the present disclosure, the gene repairoligonucleobase (GRON) is a single stranded oligodeoxynucleotidemutational vector (SSOMV), such as disclosed in International PatentApplication PCT/USOO/23457, U.S. Pat. Nos. 6,271,360, 6,479,292, and7,060,500 which is incorporated by reference in its entirety. Thesequence of the SSOMV is based on the same principles as the mutationalvectors described in U.S. Pat. Nos. 5,756,325; 5,871,984; 5,760,012;5,888,983; 5,795,972; 5,780,296; 5,945,339; 6,004,804; and 6,010,907 andin International Publication Nos. WO 98/49350; WO 99/07865; WO 99/58723;WO 99/58702; and WO 99/40789. The sequence of the SSOMV contains tworegions that are homologous with the target sequence separated by aregion that contains the desired genetic alteration termed the mutatorregion. The mutator region can have a sequence that is the same lengthas the sequence that separates the homologous regions in the targetsequence, but having a different sequence. Such a mutator region cancause a substitution. Alternatively, the homologous regions in the SSOMVcan be contiguous to each other, while the regions in the target genehaving the same sequence are separated by one, two or more nucleotides.Such an SSOMV causes a deletion from the target gene of the nucleotidesthat are absent from the SSOMV. Lastly, the sequence of the target genethat is identical to the homologous regions may be adjacent in thetarget gene but separated by one, two, or more nucleotides in thesequence of the SSOMV. Such an SSOMV causes an insertion in the sequenceof the target gene. In certain embodiments, a SSOMV does not anneal toitself.

The nucleotides of the SSOMV are deoxyribonucleotides that are linked byunmodified phosphodiester bonds except that the 3′ terminal and/or 5′terminal internucleotide linkage or alternatively the two 3′ terminaland/or 5′ terminal internucleotide linkages can be a phosphorothioate orphosphoamidate. As used herein an internucleotide linkage is the linkagebetween nucleotides of the SSOMV and does not include the linkagebetween the 3′ end nucleotide or 5′ end nucleotide and a blockingsubstituent. In a specific embodiment the length of the SSOMV is between21 and 55 deoxynucleotides and the lengths of the homology regions are,accordingly, a total length of at least 20 deoxynucleotides and at leasttwo homology regions should each have lengths of at least 8deoxynucleotides.

The SSOMV can be designed to be complementary to either the coding orthe non-coding strand of the target gene. When the desired mutation is asubstitution of a single base, it is preferred that both the mutatornucleotide and the targeted nucleotide be a pyrimidine. To the extentthat is consistent with achieving the desired functional result, it ispreferred that both the mutator nucleotide and the targeted nucleotidein the complementary strand be pyrimidines. Particularly preferred areSSOMVs that encode transversion mutations, i.e., a C or T mutatornucleotide is mismatched, respectively, with a C or T nucleotide in thecomplementary strand.

Okazaki Fragment/2′-OME GRON Design. In various embodiments, a GRON mayhave both RNA and DNA nucleotides and/or other types of nucleobases. Insome embodiments, one or more of the DNA or RNA nucleotides comprise amodification. In certain embodiments, the first 5′ nucleotide is an RNAnucleotide and the remainder of the nucleotides are DNA. In stillfurther embodiments, the first 5′ RNA nucleotide is modified with a2-O-Me. In other embodiments, the first two, three, four, five, six,seven, eight, nine, ten or more 5′ nucleotides are an RNA nucleotide andthe remainder of the nucleotides are DNA. In still further embodiments,one or more of the first two, three, four, five, six, seven, eight,nine, ten or more 5′ RNA nucleotide are modified with a 2-O-Me. In plantcells, double-strand beaks in DNA are typically repaired by the NHEJ DNArepair pathway. This pathway does not require a template to repair theDNA and is therefore error prone. The advantage of using this pathway torepair DNA for a plant cell is that it is quick, ubiquitous and mostimportantly can occur at times when a cell is not undergoing DNAreplication. Another DNA repair pathway that functions in repairingdouble-strand breaks outside of the replication fork in plant cells iscalled homologous recombination (HR); however, unlike the NHEJ pathwaythis type of repair is precise and requires the use of a DNA template(GRON). Since these GRONs mimic Okazaki fragments at the DNA replicationfork of targeted genes, it is not obvious to use them with adouble-strand DNA cutter to those skilled in the art.

Improving Efficiency

The present disclosure provides a number of approaches to increase theeffectiveness of conversion of a target gene using repairoligonucleotides, and which may be used alone or in combination with oneanother. These include:

-   1. Introducing modifications to the repair oligonucleotides which    attract DNA repair machinery to the targeted (mismatch) site.    -   A. Introduction of one or more abasic sites in the        oligonucleotide (e.g., within 10 bases, and in some embodiments        with 5 bases of the desired mismatch site) generates a lesion        which is an intermediate in base excision repair (BER), and        which attracts BER machinery to the vicinity of the site        targeted for conversion by the repair oligonucleotide. dSpacer        (abasic furan) modified oligonucleotides may be prepared as        described in, for example, Takeshita et al., J. Biol. Chem.,        262:10171-79, 1987.    -   B. Inclusion of compounds which induce single or double strand        breaks, either into the oligonucleotide or together with the        oligonucleotide, generates a lesion which is repaired by NHEJ,        microhomology-mediated end joining (MMEJ), and homologous        recombination. By way of example, the bleomycin family of        antibiotics, zinc fingers, FokI (or any type IIS class of        restriction enzyme) and other nucleases may be covalently        coupled to the 3′ or 5′ end of repair oligonucleotides, in order        to introduce double strand breaks in the vicinity of the site        targeted for conversion by the repair oligonucleotide. The        bleomycin family of antibiotics are DNA cleaving glycopeptides        which include bleomycin, zeocin, phleomycin, tallysomycin,        pepleomycin and others.    -   C. Introduction of one or more 8′oxo dA or dG incorporated in        the oligonucleotide (e.g., within 10 bases, and in some        embodiments with 5 bases of the desired mismatch site) generates        a lesion which is similar to lesions created by reactive oxygen        species. These lesions induce the so-called “pushing repair”        system. See, e.g., Kim et al., J. Biochem. Mol. Biol. 37:657-62,        2004.-   2. Increase stability of the repair oligonucleotides:    -   Introduction of a reverse base (idC) at the 3′ end of the        oligonucleotide to create a 3′ blocked end on the repair        oligonucleotide.    -   Introduction of one or more 2′O-methyl nucleotides or bases        which increase hybridization energy (see, e.g., WO2007/073149)        at the 5′ and/or 3′ of the repair oligonucleotide.    -   Introduction of one or a plurality of 2′O-methyl RNA nucleotides        at the 5′ end of the repair oligonucleotide, leading into DNA        bases which provide the desired mismatch site, thereby creating        an Okazaki Fragment-like nucleic acid structure.    -   Conjugated (5′ or 3′) intercalating dyes such as acridine,        psoralen, ethidium bromide and Syber stains.    -   Introduction of a 5′ terminus cap such as a T/A clamp, a        cholesterol moiety, SIMA (HEX), riboC and amidite.    -   Backbone modifications such as phosphothioate, 2′-O methyl,        methyl phosphonates, locked nucleic acid (LNA), MOE        (methoxyethyl), di PS and peptide nucleic acid (PNA).    -   Crosslinking of the repair oligonucleotide. e.g., with        intrastrand crosslinking reagents agents such as cisplatin and        mitomycin C.    -   Conjugation with fluorescent dyes such as Cy3, DY547, Cy3.5,        Cy3B, Cy5 and DY647.-   3. Increase hybridization energy of the repair oligonucleotide    through incorporation of bases which increase hybridization energy    (see, e.g., WO2007/073149).-   4. Increase the quality of repair oligonucleotide synthesis by using    nucleotide multimers (dimers, trimers, tetramers, etc.) as building    blocks for synthesis. This results in fewer coupling steps and    easier separation of the full length products from building blocks.-   5. Use of long repair oligonucleotides (i.e., greater than 55    nucleotides in length, for example such as the lengths described    herein, for example having one or more mutations or two or more    mutations targeted in the repair oligonucleotide.

Examples of the foregoing approaches are provided in Table 1.

TABLE 1 Exemplary GRON chemistries. Oligo type Modifications 5' mods T/Aclamp T/A clamp Backbone Phosphothioate PS modifications Intercalatingdyes 5' Acridine 3' idC Acridine, idC 2'-O-methyl DNA/RNA Cy3replacements DY547 Facilitators 2'-O-Me oligos designed 2'-O-Me 5' and3' of the converting oligo Abasic Abasic site placed in Abasic 2 variouslocations 5' and 3' to the converting base. 44 mer Assist Assistapproach Cy3, idC on one, Overlap: none on the other: 2 oligos: 1 withCy3/idC, 1 unmodified repair oligo Assist Assist approach only make theNo overlap: unmodified oligo 2 oligos: 1 with Cy3/idC, 1 unmodifiedrepair oligo Abasic THF site placed in various Tetrahydrofuran locations5' and 3' to the (dspacer) converting base. 44 mer Backbone 2'-O-Memodifications Trimers Trimer amidites, Cy3, idC Pushing repair 8'oxo dA,5' Cy3, idC Pushing repair 8'oxo dA, 5' Cy3, idC Double Bleomycin StrandBreak Crosslinker Cisplatin Crosslinker Mitomycin C Facilitators superbases 5' and 3' 2 amino dA and of converting oligo 2- thio T Superoligos 2'amino d, 5' Cy3, idC Super oligos 2-thio T, 5' Cy3, idC Superoligos 7-deaza A, 5' Cy3, idC Super oligos 7-deaza G, 5' Cy3, idC Superoligos propanyl dC, 5' Cy3, idC Intercalating dyes 5' Psoralen/3' idCPsoralen, idC Intercalating dyes 5' Ethidium bromide Ethidium bromideIntercalating dyes 5' Syber stains Syber stains 5' mods 5' Chol/3' idCCholesterol Double mutation Long oligo (55+ bases) Any modification w/ 2mutation 5' mods 5' SIMA HEX/3'idC SIMA HEX, idC Backbone 9 Methylmodifications phosphonates Backbone LNA modifications Backbone 1 MOEmodifications (methoxyethyl) Cy3 replacements Cy3.5 Cy3 replacements Cy5Backbone di PS modifications 5' mods riboC for branch nm Backbone PNAmodifications Cy3 replacements DY647 5' mods 5' branch symmetric branchamidite/idC

The foregoing modifications may also include known nucleotidemodifications such as methylation, 5′ intercalating dyes, modificationsto the 5′ and 3′ ends, backbone modifications, crosslinkers, cyclizationand ‘caps’ and substitution of one or more of the naturally occurringnucleotides with an analog such as inosine. Modifications of nucleotidesinclude the addition of acridine, amine, biotin, cascade blue,cholesterol, Cy3@, Cy5@, Cy5.5@ Daboyl, digoxigenin, dinitrophenyl,Edans, 6-FAM, fluorescein, 3′-glyceryl, HEX, IRD-700, IRD-800, JOE,phosphate psoralen, rhodamine, ROX, thiol (SH), spacers, TAMRA, TET,AMCA-S″, SE, BODIPY^(∘), Marina Blue@, Pacific Blue@, Oregon Green@,Rhodamine Green@, Rhodamine Red@, Rhodol Green@ and Texas Red@.Polynucleotide backbone modifications include methylphosphonate,2′-OMe-methylphosphonate RNA, phosphorothiorate, RNA, 2′-OMeRNA. Basemodifications include 2-amino-dA, 2-aminopurine, 3′-(ddA), 3′dA(cordycepin), 7-deaza-dA, 8-Br-dA, 8-oxo-dA, N6-Me-dA, abasic site(dSpacer), biotin dT, 2′-OMe-5Me-C, 2′-OMe-propynyl-C, 3′-(5-Me-dC),3′-(ddC), 5-Br-dC, 5-1-duc, 5-Me-dC, 5-F-dC, carboxy-dT, convertible dA,convertible dC, convertible dG, convertible dT, convertible dU,7-deaza-dG, 8-Br-dG, 8-oxo-dG, O6-Me-dG, S6-DNP-dG, 4-methyl-indole,5-nitroindole, 2′-OMe-inosine, 2′-dl, o6-phenyl-dl, 4-methyl-indole,2′-deoxynebularine, 5-nitroindole, 2-aminopurine, dP (purine analogue),dK (pyrimidine analogue), 3-nitropyrrole, 2-thio-dT, 4-thio-dT,biotin-dT, carboxy-dT, 04-Me-dT, 04-triazol dT, 2′-OMe-propynyl-U,5-Br-dU, 2′-dU, 5-F-dU, 5-1-dU, 04-triazol dU. Said terms also encompasspeptide nucleic acids (PNAs), a DNA analogue in which the backbone is apseudopeptide consisting of N-(2-aminoethyl)-glycine units rather than asugar. PNAs mimic the behavior of DNA and bind complementary nucleicacid strands. The neutral backbone of PNA results in stronger bindingand greater specificity than normally achieved. In addition, the uniquechemical, physical and biological properties of PNA have been exploitedto produce powerful biomolecular tools, antisense and antigene agents,molecular probes and biosensors.

Oligonucleobases may have nick(s), gap(s), modified nucleotides such asmodified oligonucleotide backbones, abasic nucleotides, or otherchemical moieties. In a further embodiment, at least one strand of theoligonucleobase includes at least one additional modified nucleotide,e.g., a 2′-O-methyl modified nucleotide such, a MOE (methoxyethyl), anucleotide having a 5′-phosphorothioate group, a terminal nucleotidelinked to a cholesteryl derivative, a 2′-deoxy-2′-fluoro modifiednucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, anabasic nucleotide (the nucleobase is missing or has a hydroxyl group inplace thereof (see, e.g., Glen Research,www.glenresearch.com/GlenReports/GR21-14), a 2′-amino-modifiednucleotide, a 2′-alkyl-modified nucleotide, a morpholino nucleotide, aphosphoramidite, and a non-natural base comprising nucleotide. Varioussalts, mixed salts and free acid forms are also included.

Preferred modified oligonucleotide backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphoro-dithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates, 5′-alkylenephosphonates and chiral phosphonates, phosphinates, phosphoramidatesincluding 3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkyl-phosphonates,thionoalkylphosphotriesters, selenophosphates and boranophosphateshaving normal 3′-5′ linkages, 2′-5′ linked analogs of these, and thosehaving inverted polarity wherein one or more internucleotide linkages isa 3′ to 3′,5 ′ to 5′ or 2′ to 2′ linkage. Preferred oligonucleotideshaving inverted polarity comprise a single 3′ to 3′ linkage at the3′-most internucleotide linkage i.e. a single inverted nucleosideresidue which may be abasic (the nucleobase is missing or has a hydroxylgroup in place thereof). The most common use of a linkage inversion isto add a 3′-3′ linkage to the end of an antisense oligonucleotide with aphosphorothioate backbone. The 3′-3′ linkage further stabilizes theantisense oligonucleotide to exonuclease degradation by creating anoligonucleotide with two 5′-OH ends and no 3′-OH end. Linkage inversionscan be introduced into specific locations during oligonucleotidesynthesis through use of “reversed phosphoramidites”. These reagentshave the phosphoramidite groups on the 5′-OH position and thedimethoxytrityl (DMT) protecting group on the 3′-OH position. Normally,the DMT protecting group is on the 5′-OH and the phosphoramidite is onthe 3′-OH.

Examples of modified bases include, but are not limited to,2-aminopurine, 2′-amino-butyryl pyrene-uridine, 2′-aminouridine,2′-deoxyuridine, 2′-fluoro-cytidine, 2′-fluoro-uridine,2,6-diaminopurine, 4-thio-uridine, 5-bromo-uridine, 5-fluoro-cytidine,5-fluorouridine, 5-indo-uridine, 5-methyl-cytidine, inosine,N3-methyl-uridine, 7-deaza-guanine, 8-aminohexyl-amino-adenine,6-thio-guanine, 4-thio-thymine, 2-thio-thymine, 5-iodo-uridine,5-iodo-cytidine, 8-bromo-guanine, 8-bromo-adenine, 7-deaza-adenine,7-diaza-guanine, 8-oxo-guanine, 5,6-dihydro-uridine, and5-hydroxymethyl-uridine. These synthetic units are commerciallyavailable; (for example, purchased from Glen Research Company) and canbe incorporated into DNA by chemical synthesis.

Examples of modification of the sugar moiety are 3′-deoxylation,2′-fluorination, and arabanosidation, however, it is not to be construedas being limited thereto. Incorporation of these into DNA is alsopossible by chemical synthesis.

Examples of the 5′ end modification are 5′-amination, 5′-biotinylation,5′-fluoresceinylation, 5′-tetrafluoro-fluoreceinyaltion, 5′-thionation,and 5′-dabsylation, however it is not to be construed as being limitedthereto.

Examples of the 3′ end modification are 3′-amination, 3′-biotinylation,2,3-dideoxidation, 3′-thionation, 3′-dabsylation, 3′-carboxylation, and3′-cholesterylation, however, it is not to be construed as being limitedthereto.

In one preferred embodiment, the oligonucleobase can contain a 5′blocking substituent that is attached to the 5′ terminal carbons througha linker. The chemistry of the linker is not critical other than itslength, which should in some embodiments be at least 6 atoms long andthat the linker should be flexible. A variety of non-toxic substituentssuch as biotin, cholesterol or other steroids or a non-intercalatingcationic fluorescent dye can be used. Particularly preferred reagents tomake oligonucleobases are the reagents sold as Cy3™ and Cy5™ by GlenResearch, Sterling Va. (now GE Healthcare), which are blockedphosphoroamidites that upon incorporation into an oligonucleotide yield3,3,3′,3 ′-tetramethyl N,N′-isopropyl substituted indomonocarbocyanineand indodicarbocyanine dyes, respectively. Cy3 is particularlypreferred. When the indocarbocyanine is N-oxyalkyl substituted it can beconveniently linked to the 5′ terminal of the oligodeoxynucleotide as aphosphodiester with a 5′ terminal phosphate. When the commerciallyavailable Cy3 phosphoramidite is used as directed, the resulting 5′modification consists of a blocking substituent and linker togetherwhich are a N-hydroxypropyl, N′-phosphatidylpropyl 3,3,3′,3′-tetramethyl indomonocarbocyanine. Other dyes contemplated includeRhodamine6G, Tetramethylrhodamine, Sulforhodamine 101, Merocyanine 540,Atto565, Atto550 26, Cy3.5, Dy547, Dy548, Dy549, Dy554, Dy555, Dy556,Dy560, mStrawberry and mCherry.

In a preferred embodiment the indocarbocyanine dye is tetra substitutedat the 3 and 3′ positions of the indole rings. Without limitations as totheory these substitutions prevent the dye from being an intercalatingdye. The identity of the substituents at these positions is notcritical.

The oligo designs herein described might also be used as more efficientdonor templates in combination with other DNA editing or recombinationtechnologies including, but not limited to, gene targeting usingsite-specific homologous recombination by zinc finger nucleases,Transcription Activator-Like Effector Nucleases (TALENs) or ClusteredRegularly Interspaced Short Palindromic Repeats (CRISPRs).

The present disclosure in certain aspects and embodiments generallyrelates to methods for the efficient modification of genomic cellularDNA and/or recombination of DNA into the genomic DNA of cells. Althoughnot limited to any particular use, some methods provided herein may incertain embodiments be useful in, for example, introducing amodification into the genome of a cell for the purpose of determiningthe effect of the modification on the cell. For example, a modificationmay be introduced into the nucleotide sequence which encodes an enzymeto determine whether the modification alters the enzymatic activity ofthe enzyme, and/or determine the location of the enzyme's catalyticregion. Alternatively, the modification may be introduced into thecoding sequence of a DNA-binding protein to determine whether the DNAbinding activity of the protein is altered, and thus to delineate theparticular DNA-binding region within the protein. Yet anotheralternative is to introduce a modification into a non-coding regulatorysequence (e.g., promoter, enhancer, regulatory RNA sequence (miRNA),etc.) in order to determine the effect of the modification on the levelof expression of a second sequence which is operably linked to thenon-coding regulatory sequence. This may be desirable to, for example,define the particular sequence which possesses regulatory activity.

DNA Cutters

One strategy for producing targeted gene disruption is through thegeneration of single strand or double strand DNA breaks using a DNAcutter such as a site-specific endonuclease. Endonucleases are mostoften used for targeted gene disruption in organisms that havetraditionally been refractive to more conventional gene targetingmethods, such as algae, plants, and large animal models, includinghumans. For example, there are currently human clinical trials underwayinvolving zinc finger nucleases for the treatment and prevention of HIVinfection. Additionally, endonuclease engineering is currently beingused in attempts to disrupt genes that produce undesirable phenotypes incrops.

Zinc Fingers

One class of artificial endonucleases is the zinc finger endonucleases.Zinc finger endonucleases combine a non-specific cleavage domain,typically that of FokI endonuclease, with zinc finger protein domainsthat are engineered to bind to specific DNA sequences. The modularstructure of the zinc finger endonucleases makes them a versatileplatform for delivering site-specific double-strand breaks to thegenome. As FokI endonuclease cleaves as a dimer, one strategy to preventoff-target cleavage events has been to design zinc finger domains thatbind at adjacent 9 base pair sites. See also U.S. Pat. Nos. 7,285,416;7,521,241; 7,361,635; 7,273,923; 7,262,054; 7,220,719; 7,070,934;7,013,219; 6,979,539; 6,933,113; 6,824,978; each of which is herebyherein incorporated by reference in its entirety.

TALENs

TALENs are targetable nucleases are used to induce single- anddouble-strand breaks into specific DNA sites, which are then repaired bymechanisms that can be exploited to create sequence alterations at thecleavage site.

The fundamental building block that is used to engineer the DNA-bindingregion of TALENs is a highly conserved repeat domain derived fromnaturally occurring TALEs encoded by Xanthomonas spp. proteobacteria.DNA binding by a TALEN is mediated by arrays of highly conserved 33-35amino acid repeats that are flanked by additional TALE-derived domainsat the amino-terminal and carboxy-terminal ends of the repeats.

These TALE repeats specifically bind to a single base of DNA, theidentity of which is determined by two hypervariable residues typicallyfound at positions 12 and 13 of the repeat, with the number of repeatsin an array corresponded to the length of the desired target nucleicacid, the identity of the repeat selected to match the target nucleicacid sequence. In some embodiments, the target nucleic acid is between15 and 20 base pairs in order to maximize selectivity of the targetsite. Cleavage of the target nucleic acid typically occurs within 50base pairs of TALEN binding. Computer programs for TALEN recognitionsite design have been described in the art. See, e.g., Cermak et al.,Nucleic Acids Res. 2011 July; 39(12): e82.

Once designed to match the desired target sequence, TALENs can beexpressed recombinantly and introduced into protoplasts as exogenousproteins, or expressed from a plasmid within the protoplast oradministered as mRNA.

Meganucleases

The homing endonucleases, also known as meganucleases, are sequencespecific endonucleases that generate double strand breaks in genomic DNAwith a high degree of specificity due to their large (e.g., >14 bp)cleavage sites. While the specificity of the homing endonucleases fortheir target sites allows for precise targeting of the induced DNAbreaks, homing endonuclease cleavage sites are rare and the probabilityof finding a naturally occurring cleavage site in a targeted gene islow.

Another class of artificial endonucleases is the engineeredmeganucleases. Engineered homing endonucleases are generated bymodifying the specificity of existing homing endonucleases. In oneapproach, variations are introduced in the amino acid sequence ofnaturally occurring homing endonucleases and then the resultantengineered homing endonucleases are screened to select functionalproteins which cleave a targeted binding site. In another approach,chimeric homing endonucleases are engineered by combining therecognition sites of two different homing endonucleases to create a newrecognition site composed of a half-site of each homing endonuclease.See e.g., U.S. Pat. No. 8,338,157.

CRISPRs or CRISPR/Cas Systems

CRISPR-Cas system contains three basic design components: 1) Cas gene,transcript (e.g., mRNA) or protein; 2) guide RNA (gRNA); and 3) crRNAs(CRISPR RNA) are RNA segments processed from RNA transcripts encodingthe CRISPR repeat arrays, which harbor a “protospacer” region that arecomplementary to a foreign DNA site (e.g., endogenous DNA target region)and a part of the CRISPR repeat. See e.g., PCT Applciation NosWO/2014/093661 and WO/2013/176772.

Cas (CRISPR Associated) Gene, Transcript (e.g., mRNA) or Protein

Transient Cas expression from a plasmid vector, direct delivery of Casprotein and or direct delivery of Cas mRNA into plant cells. Cas genesare codon optimized for expression in higher plants, algae or yeast andare driven by either a constitutive, inducible, tissue-specific orspecies-specific promoter when applicable. Cas transcript terminationand polyadenlyation signals are either NosT, RBCT, HSP18.2T or othergene specific or species-specific terminators. Cas gene cassettes ormRNA may contain introns, either native or in combination withgene-specific promoters and or synthetic promoters. Cas protein maycontain one or more nuclear localization signal sequences (NLS),mutations, deletions, alterations or truncations. In transientexpression systems, Cas gene cassettes may be combined with othercomponents of the CRISPR-Cas system such as gRNA cassettes on the sametransient expression vector. Alternatively, Cas gene cassettes may belocated and expressed from constructs independent of gRNA cassettes orfrom other components of the CRISPR-Cas system. CRISPR associated (Cas)gene-encode for proteins with a variety of predicted nucleicacid-manipulating activities such as nucleases, helicases andpolymerase. Cas genes include cas9. Cas9 is a gene encoding a largeprotein containing a predicted RuvC-like and HNH endonuclease domainsand is associated with the CRISPR adaptive immunity system that ispresent in most archaea and many bacteria. Cas9 protein consists of twolobes:

-   1) Recognition (REC) lobe-consists of three domains:    -   a) BH (bridge helix)    -   b) REC1-facilitates RNA-guided DNA targeting    -   c) REC2-facilitates RNA-guided DNA targeting-   2) Nuclease (NUC) lobe-consists of three domains:    -   a) RuvC-facilitates RNA-guided DNA targeting; endonuclease        activity    -   b) HNH-endonuclease activity    -   c) PI-PAM interacting

In other embodiments, the cas gene may be a homolog of cas9 in which theRuvC, HNH, REC and BH domains are highly conserved. In some embodiments,cas genes are those from the following species.

Guide RNA (gRNA)

gRNA or sgRNA (single guide RNA) is engineered as a fusion between acrRNA and part of the transactivating CRISPR RNA (tracrRNA) sequence,which guides the Cas9 to a specific target DNA sequence that iscomplementary to the protospacer region. Guide RNA may include anexpression cassette containing a chimeric RNA design with a longtracerRNA hybrid, short tracrRNA hybrid or a native CRISPRarray+tracrRNA conformation. Chimeric gRNA combines the targetingspecificity of the crRNA with the scaffolding properties of the tracrRNAinto a single transcript. gRNA transient expression is controlled byspecies-specific higher plant RNA Polymerase III promoters such as thosefrom the U6 or U3 snRNA gene family (Wang et al 2008). gRNA transcripttermination is controlled by a 6-20 nucleotide tract of poly dT as perWang et al 2008. gRNA expression cassettes are located on the same ordifferent transient expression vectors from other components of theCRISPR-Cas system. gRNA transcripts may be synthesized in-vitro anddelivered directly into plant cells, independent of or in combinationwith gRNA transient expression vectors.

Target Region

Guide RNAs contain two components that define specificity to a DNAtarget region, a proto-spacer and a proto-spacer adjacent motif (PAM).Proto-spacer sequence, typically 20 nucleotides but can vary based onthe DNA target, provides DNA sequence specificity for the CRISPR-Cascomplex. DNA targets also contain a NNG or NAG tri-nucleotide sequence(PAM) where N denotes any nucleotide, immediately 3′ or downstream ofthe proto-spacer.

One Component Approach

Similar to Le Cong et al. (2013) and others, a simplified “one componentapproach” to CRISPR-Cas gene editing wherein a single transientexpression construct contains all components of the CRISPR-Cas complex,i.e. both the gRNA and the Cas expressions cassettes. This allows for aneasy modular design for targeting single or multiple loci in any givenplant or crop. Targeting multiple loci can be achieved by simplyswapping in the target-specific gRNA cassettes. Additionally, speciesspecific promoters, terminators or other expressing enhancing elementscan easily be shuttled in and out of “one component approach” transientvectors allowing for optimal expression of both gRNA and Cas protein ina species specific manner.

Two Component Approach

In the two component approach, Cas and gRNA expression cassettes arelocated on different transient expression vectors. This allows fordelivery of a CRISPR-Cas editing components separately, allowing fordifferent ratios of gRNA to Cas within the same cell. Similar to the onecomponent approach, the two component approach also allows forpromoters, terminators or other elements affecting expression ofCRISPR-Cas components to be easily altered and allow targeting of DNA ina species-specific manner.

Antibiotics

Another class of endonucleases are antibiotics which are DNA cleavingglycopeptides such as the bleomycin family of antibiotics are DNAcleaving glycopeptides which include bleomycin, zeocin, phleomycin,tallysomycin, pepleomycin and others which are further described herein.

Other DNA-modifying molecules may be used in targeted generecombination. For example, peptide nucleic acids may be used to inducemodifications to the genome of the target cell or cells (see. e.g.,Ecker, U.S. Pat. No. 5,986,053 herein incorporated by reference). Inbrief, synthetic nucleotides comprising, at least, a partial peptidebackbone are used to target a homologous genomic nucleotide sequence.Upon binding to the double-helical DNA, or through a mutagen ligated tothe peptide nucleic acid, modification of the target DNA sequence and/orrecombination is induced to take place. Targeting specificity isdetermined by the degree of sequence homology between the targetingsequence and the genomic sequence.

Furthermore, the present disclosure is not limited to the particularmethods which are used herein to execute modification of genomicsequences. Indeed, a number of methods are contemplated. For example,genes may be targeted using triple helix forming oligonucleotides (TFO).TFOs may be generated synthetically, for example, by PCR or by use of agene synthesizer apparatus. Additionally, TFOs may be isolated fromgenomic DNA if suitable natural sequences are found. TFOs may be used ina number of ways, including, for example, by tethering to a mutagen suchas, but not limited to, psoralen or chlorambucil (see, e.g., Havre etal., Proc Nat'l Acad Sci, U.S.A. 90:7879-7883, 1993; Havre et al., JVirol 67:7323-7331, 1993; Wang et al., Mol Cell Biol 15:1759-1768, 1995;Takasugi et al., Proc Nat'l Acad Sci. U.S.A. 88:5602-5606, 1991;Belousov et al., Nucleic Acids Res 25:3440-3444, 1997). Furthermore, forexample, TFOs may be tethered to donor duplex DNA (see, e.g., Chan etal., J Biol Chem 272:11541-11548, 1999). TFOs can also act by bindingwith sufficient affinity to provoke error-prone repair (Wang et al.,Science 271:802-805, 1996).

The methods disclosed herein are not necessarily limited to the natureor type of DNA-modifying reagent which is used. For example, suchDNA-modifying reagents release radicals which result in DNA strandbreakage. Alternatively, the reagents alkylate DNA to form adducts whichwould block replication and transcription. In another alternative, thereagents generate crosslinks or molecules that inhibit cellular enzymesleading to strand breaks. Examples of DNA-modifying reagents which havebeen linked to oligonucleotides to form TFOs include, but are notlimited to, indolocarbazoles, napthalene diimide (NDI), transplatin,bleomycin, analogues of cyclopropapyrroloindole, andphenanthodihydrodioxins. In particular, indolocarbazoles aretopoisomerase I inhibitors. Inhibition of these enzymes results instrand breaks and DNA protein adduct formation (Arimondo et al.,Bioorganic and Medicinal Chem. 8, 777, 2000). NDI is a photooxidant thatcan oxidize guanines which could cause mutations at sites of guanineresidues (Nunez, et al., Biochemistry, 39, 6190, 2000). Transplatin hasbeen shown to react with DNA in a triplex target when the TFO is linkedto the reagent. This reaction causes the formation of DNA adducts whichwould be mutagenic (Columbier, et al., Nucleic Acids Research, 24: 4519,1996). Bleomycin is a DNA breaker, widely used as a radiation mimetic.It has been linked to oligonucleotides and shown to be active as abreaker in that format (Sergeyev, Nucleic Acids Research 23, 4400, 1995;Kane, et al., Biochemistry, 34, 16715, 1995). Analogues ofcyclopropapyrroloindole have been linked to TFOs and shown to alkylateDNA in a triplex target sequence. The alkylated DNA would then containchemical adducts which would be mutagenic (Lukhtanov, et al., NucleicAcids Research, 25, 5077, 1997). Phenanthodihydrodioxins are maskedquinones that release radical species upon photoactivation. They havebeen linked to TFOs and have been shown to introduce breaks into duplexDNA on photoactivation (Bendinskas et al., Bioconjugate Chem. 9, 555,1998).

Other methods of inducing modifications and/or recombination arecontemplated by the present disclosure. For example, another embodimentinvolves the induction of homologous recombination between an exogenousDNA fragment and the targeted gene (see e.g., Capecchi et al., Science244:1288-1292, 1989) or by using peptide nucleic acids (PNA) withaffinity for the targeted site. Still other methods include sequencespecific DNA recognition and targeting by polyamides (see e.g., Dervanet al., Curr Opin Chem Biol 3:688-693, 1999; Biochemistry 38:2143-2151,1999) and the use nucleases with site specific activity (e.g., zincfinger proteins, TALENs, Meganucleases and/or CRISPRs).

The present disclosure is not limited to any particular frequency ofmodification and/or recombination. In some embodiments the methodsdisclosed herein result in a frequency of modification in the targetnucleotide sequence of from 0.2% to 3%. Nonetheless, any frequency(i.e., between 0% and 100%) of modification and/or recombination iscontemplated to be within the scope of the present disclosure. Thefrequency of modification and/or recombination is dependent on themethod used to induce the modification and/or recombination, the celltype used, the specific gene targeted and the DNA mutating reagent used,if any. Additionally, the method used to detect the modification and/orrecombination, due to limitations in the detection method, may notdetect all occurrences of modification and/or recombination.Furthermore, some modification and/or recombination events may besilent, giving no detectable indication that the modification and/orrecombination has taken place. The inability to detect silentmodification and/or recombination events gives an artificially lowestimate of modification and/or recombination. Because of these reasons,and others, the disclosure is not necessarily limited to any particularmodification and/or recombination frequency. In one embodiment, thefrequency of modification and/or recombination is between 0.01% and100%. In another embodiment, the frequency of modification and/orrecombination is between 0.01% and 50%. In yet another embodiment, thefrequency of modification and/or recombination is between 0.1% and 10%.In still yet another embodiment, the frequency of modification and/orrecombination is between 0.1% and 5%.

The term “frequency of mutation” as used herein in reference to apopulation of cells which are treated with a DNA-modifying molecule thatis capable of introducing a mutation into a target site in the cells'genome, refers to the number of cells in the treated population whichcontain the mutation at the target site as compared to the total numberof cells which are treated with the DNA-modifying molecule. For example,with respect to a population of cells which is treated with theDNA-modifying molecule TFO tethered to psoralen which is designed tointroduce a mutation at a target site in the cells' genome, a frequencyof mutation of 5% means that of a total of 100 cells which are treatedwith TFO-psoralen, 5 cells contain a mutation at the target site.

Although the present disclosure is not necessarily limited to any degreeof precision in the modification and/or recombination of DNA in thecell, it is contemplated that some embodiments of the present disclosurerequire higher degrees of precision, depending on the desired result.For example, the specific sequence changes required for gene repair(e.g., particular base changes) require a higher degree of precision ascompared to producing a gene knockout wherein only the disruption of thegene is necessary. With the methods of the present disclosure,achievement of higher levels of precision in modification and/orhomologous recombination techniques is greater than with prior artmethods.

Delivery of Gene Repair Oligonucleobases into Plant Cells

Any commonly known method used to transform a plant cell can be used fordelivering the gene repair oligonucleobases. Illustrative methods arelisted below. The methods and compositions herein may involve any ofmany methods to transfect the cells with the DNA-modifying reagent orreagents. Methods for the introduction of DNA modifying reagents into acell or cells are well known in the art and include, but are not limitedto, microinjection, electroporation, passive adsorption, calciumphosphate-DNA co-precipitation. DEAE-dextran-mediated transfection,polybrene-mediated transfection, liposome fusion, lipofectin,nucleofection, protoplast fusion, retroviral infection, biolistics(i.e., particle bombardment) and the like.

The use of metallic microcarriers (microspheres) for introducing largefragments of DNA into plant cells having cellulose cell walls byprojectile penetration is well known to those skilled in the relevantart (henceforth biolistic delivery). U.S. Pat. Nos. 4,945,050; 5,100,792and 5,204,253 describe general techniques for selecting microcarriersand devices for projecting them.

Specific conditions for using microcarriers in the methods disclosedherein may include the conditions described in International PublicationWO 99/07865. In an illustrative technique, ice cold microcarriers (60mg/mL), mixed duplex oligonucleotide (60 mg/mL) 2.5 M CaCl₂) and 0.1 Mspermidine are added in that order, the mixture gently agitated, e.g.,by vortexing, for 10 minutes and then left at room temperature for 10minutes, whereupon the microcarriers are diluted in 5 volumes ofethanol, centrifuged and resuspended in 100% ethanol. Good results canbe obtained with a concentration in the adhering solution of 8-10 μg/μLmicrocarriers, 14-17 μg/mL mixed duplex oligonucleotide, 1.1-1.4 M CaCl₂and 18-22 mM spermidine. Optimal results were observed under theconditions of 8 μg/μL microcarriers, 16.5 gig/mL mixed duplexoligonucleotide, 1.3 M CaCl₂ and 21 mM spermidine.

Gene repair oligonucleobases can also be introduced into plant cellsusing microfibers to penetrate the cell wall and cell membrane. U.S.Pat. No. 5,302,523 to Coffee et al describes the use of silicon carbidefibers to facilitate transformation of suspension maize cultures ofBlack Mexican Sweet. Any mechanical technique that can be used tointroduce DNA for transformation of a plant cell using microfibers canbe used to deliver gene repair oligonucleobases for transmutation.

An illustrative technique for microfiber delivery of a gene repairoligonucleobase is as follows: Sterile microfibers (2 μg) are suspendedin 150 μL of plant culture medium containing about 10 μg of a mixedduplex oligonucleotide. A suspension culture is allowed to settle andequal volumes of packed cells and the sterile fiber/nucleotidesuspension are vortexed for 10 minutes and plated. Selective media areapplied immediately or with a delay of up to about 120 h as isappropriate for the particular trait.

In an alternative embodiment, the gene repair oligonucleobases can bedelivered to the plant cell by electroporation of a protoplast derivedfrom a plant part. The protoplasts are formed by enzymatic treatment ofa plant part, particularly a leaf, according to techniques well known tothose skilled in the art. See, e.g., Gallois et al, 1996, in Methods inMolecular Biology 55:89-107, Humana Press, Totowa, N.J.; Kipp et al.,1999, in Methods in Molecular Biology 133:213-221. Humana Press. Totowa,N.J. The protoplasts need not be cultured in growth media prior toelectroporation. Illustrative conditions for electroporation are 300,000protoplasts in a total volume of 0.3 mL with a concentration of generepair oligonucleobase of between 0.6-4 μg/mL.

In an alternative embodiment, nucleic acids are taken up by plantprotoplasts in the presence of the membrane-modifying agent polyethyleneglycol, according to techniques well known to those skilled in the art.In another alternative embodiment, the gene repair oligonucleobases canbe delivered by injecting it with a microcapillary into plant cells orinto protoplasts.

In an alternative embodiment, nucleic acids are embedded in microbeadscomposed of calcium alginate and taken up by plant protoplasts in thepresence of the membrane-modifying agent polyethylene glycol (see, e.g.,Sone et al., 2002, Liu et al., 2004).

In an alternative embodiment, nucleic acids frozen in water andintroduced into plant cells by bombardment in the form of microparticles(see, e.g., Gilmore, 1991, U.S. Pat. No. 5,219,746; Brinegar et al.).

In an alternative embodiment, nucleic acids attached to nanoparticlesare introduced into intact plant cells by incubation of the cells in asuspension containing the nanoparticle (see, e.g., Pasupathy et al.,2008) or by delivering them into intact cells through particlebombardment or into protoplasts by co-incubation (see. e.g., Torney etal., 2007).

In an alternative embodiment, nucleic acids complexed with penetratingpeptides and delivered into cells by co-incubation (see, e.g., Chugh etal., 2008, WO 2008148223 A1; Eudes and Chugh).

In an alternative embodiment, nucleic acids are introduced into intactcells through electroporation (see. e.g., He et al., 1998. US2003/0115641 A1, Dobres et al.).

In an alternative embodiment, nucleic acids are delivered into cells ofdry embryos by soaking them in a solution with nucleic acids (see, e.g.,Töpfer et al., 1989, Senaratna et al., 1991) or in other embodiments areintroduced by Cellsqueeze (SQZ Biotech).

Selection of Plants

In various embodiments, plants as disclosed herein can be of any speciesof dicotyledonous, monocotyledonous or gymnospermous plant, includingany woody plant species that grows as a tree or shrub, any herbaceousspecies, or any species that produces edible fruits, seeds orvegetables, or any species that produces colorful or aromatic flowers.For example, the plant maybe selected from a species of plant from thegroup consisting of canola, sunflower, corn, tobacco, sugar beet,cotton, maize, wheat, barley, rice, alfalfa, barley, sorghum, tomato,mango, peach, apple, pear, strawberry, banana, melon, cassava, potato,carrot, lettuce, onion, soy bean, soya spp, sugar cane, pea, chickpea,field pea, fava bean, lentils, turnip, rutabaga, brussel sprouts, lupin,cauliflower, kale, field beans, poplar, pine, eucalyptus, grape, citrus,triticale, alfalfa, rye, oats, turf and forage grasses, flax, oilseedrape, mustard, cucumber, morning glory, balsam, pepper, eggplant,marigold, lotus, cabbage, daisy, carnation, tulip, iris, lily, and nutproducing plants insofar as they are not already specifically mentioned.

Plants and plant cells can be tested for resistance or tolerance to anherbicide using commonly known methods in the art, e.g., by growing theplant or plant cell in the presence of an herbicide and measuring therate of growth as compared to the growth rate in the absence of theherbicide.

As used herein, substantially normal growth of a plant, plant organ,plant tissue or plant cell is defined as a growth rate or rate of celldivision of the plant, plant organ, plant tissue, or plant cell that isat least 35%, at least 50%, at least 60%, or at least 75% of the growthrate or rate of cell division in a corresponding plant, plant organ,plant tissue or plant cell expressing the wild-type protein of interest.

As used herein, substantially normal development of a plant, plantorgan, plant tissue or plant cell is defined as the occurrence of one ormore development events in the plant, plant organ, plant tissue or plantcell that are substantially the same as those occurring in acorresponding plant, plant organ, plant tissue or plant cell expressingthe wild-type protein.

In certain embodiments plant organs provided herein include, but are notlimited to, leaves, stems, roots, vegetative buds, floral buds,meristems, embryos, cotyledons, endosperm, sepals, petals, pistils,carpels, stamens, anthers, microspores, pollen, pollen tubes, ovules,ovaries and fruits, or sections, slices or discs taken therefrom. Planttissues include, but are not limited to, callus tissues, ground tissues,vascular tissues, storage tissues, meristematic tissues, leaf tissues,shoot tissues, root tissues, gall tissues, plant tumor tissues, andreproductive tissues. Plant cells include, but are not limited to,isolated cells with cell walls, variously sized aggregates thereof, andprotoplasts.

Plants are substantially “tolerant” to a relevant herbicide when theyare subjected to it and provide a dose/response curve which is shiftedto the right when compared with that provided by similarly subjectednon-tolerant like plant. Such dose/response curves have “dose” plottedon the X-axis and “percentage kill”, “herbicidal effect”, etc., plottedon the y-axis. Tolerant plants will require more herbicide thannon-tolerant like plants in order to produce a given herbicidal effect.Plants that are substantially “resistant” to the herbicide exhibit few,if any, necrotic, lytic, chlorotic or other lesions, when subjected toherbicide at concentrations and rates which are typically employed bythe agrochemical community to kill weeds in the field. Plants which areresistant to an herbicide are also tolerant of the herbicide.

Generation of Plants

Tissue culture of various tissues of plant species and regeneration ofplants therefrom is known. For example, the propagation of a canolacultivar by tissue culture is described in any of the following but notlimited to any of the following: Chuong et al., “A Simple Culture Methodfor Brassica hypocotyls Protoplasts,” Plant Cell Reports 4:4-6, 1985;Barsby, T. L., et al., “A Rapid and Efficient Alternative Procedure forthe Regeneration of Plants from Hypocotyl Protoplasts of Brassicanapus,” Plant Cell Reports (Spring, 1996); Kartha, K., et al., “In vitroPlant Formation from Stem Explants of Rape,” Physiol. Plant, 31:217-220,1974; Narasimhulu, S., et al., “Species Specific Shoot RegenerationResponse of Cotyledonary Explants of Brassicas,” Plant Cell Reports(Spring 1988); Swanson, E., “Microspore Culture in Brassica,” Methods inMolecular Biology, Vol. 6, Chapter 17, p. 159, 1990.

Further reproduction of the variety can occur by tissue culture andregeneration. Tissue culture of various tissues of soybeans andregeneration of plants therefrom is well known and widely published. Forexample, reference may be had to Komatsuda, T. et al., “Genotype XSucrose Interactions for Somatic Embryogenesis in Soybeans,” Crop Sci.31:333-337, 1991; Stephens, P. A., et al., “Agronomic Evaluation ofTissue-Culture-Derived Soybean Plants,” Theor. Appl. Genet. 82:633-635,1991; Komatsuda, T. et al., “Maturation and Germination of SomaticEmbryos as Affected by Sucrose and Plant Growth Regulators in SoybeansGlycine gracilis Skvortz and Glycine max (L.) Merr.” Plant Cell, Tissueand Organ Culture, 28:103-113, 1992; Dhir, S. et al., “Regeneration ofFertile Plants from Protoplasts of Soybean (Glycine max L. Merr.);Genotypic Differences in Culture Response,” Plant Cell Reports11:285-289, 1992; Pandey, P. et al., “Plant Regeneration from Leaf andHypocotyl Explants of Glycine wightii (W. and A.) VERDC. var.longicauda,” Japan J. Breed. 42:1-5, 1992; and Shetty, K., et al.,“Stimulation of In Vitro Shoot Organogenesis in Glycine max (Merrill.)by Allantoin and Amides,” Plant Science 81:245-251, 1992. Thedisclosures of U.S. Pat. No. 5,024,944 issued Jun. 18, 1991 to Collinset al., and U.S. Pat. No. 5,008,200 issued Apr. 16, 1991 to Ranch etal., are hereby incorporated herein in their entirety by reference.

EXEMPLARY EMBODIMENTS

In addition to the aspects and embodiments described and providedelsewhere in this disclosure, the following non-limiting list ofparticular embodiments are specifically contemplated.

1. A method of causing a genetic change in a cell, said methodcomprising exposing said cell to a DNA cutter and a modified GRON.

2. A cell comprising a DNA cutter and a GRON.

3. The method or cell of any of the preceding embodiments, wherein saidcells is one or more species of cell selected from the group consistingof plant, bacteria, yeast, fungi, algae, and mammalian.

4. The method or cell of any of the preceding embodiments, wherein saidcells is one or more species of cell selected from the group consistingof Escherichia coli, Mycobacterium smegmatis, Baccillus subtilis,Chlorella, Bacillus thuringiensis, Saccharomyces cerevisiae, Yarrowialipolytica, Chlamydamonas rhienhardtii, Pichia pastoris,Corynebacterium, Aspergillus niger, and Neurospora crassa. Arabidopsisthaliana, Solanum tuberosum, Solanum phureja, Oryza sativa, Glycine max,Amaranthus tuberculatus, Linum usitatissimum, and Zea mays5. The method or cell of any of the preceding embodiments, wherein saidcell is Yarrowia lipolytica.6. The method or cell of any of the preceding embodiments, wherein saidcell is a yeast cell that is not Saccharomyces cerevisiae.7. A method of causing a genetic change in a plant cell, said methodcomprising exposing said cell to a DNA cutter and a modified GRON.8. A plant cell comprising a DNA cutter and a modified GRON.9. A method of causing a genetic change in a plant cell, said methodcomprising exposing said cell to a DNA cutter and a GRON that comprisesDNA and/or RNA.10. A plant cell comprising a DNA cutter that comprises DNA and/or RNAand/or protein.11. A method of causing a genetic change in a Acetyl-Coenzyme Acarboxylase (ACCase) gene in a cell, wherein said genetic change causesa change in the Acetyl-Coenzyme A carboxylase (ACCase) protein at one ormore amino acid positions, said positions selected from the groupconsisting of 1781, 1783, 1786, 2078, 2079, 2080 and 2088 based on thenumbering of the blackgrass reference sequence SEQ ID NO:1 or at ananalogous amino acid residue in an ACCase paralog said method comprisingexposing said cell to a modified GRON.12. A method of causing a genetic change in a Acetyl-Coenzyme Acarboxylase (ACCase) gene in a cell, wherein said genetic change causesa change in the Acetyl-Coenzyme A carboxylase (ACCase) protein at one ormore amino acid positions, said positions selected from the groupconsisting of 1781, 1783, 1786, 2078, 2079, 2080 and 2088 based on thenumbering of the blackgrass reference sequence SEQ ID NO:1 or at ananalogous amino acid residue in an ACCase paralog said method comprisingexposing said cell to a DNA cutter and a modified GRON.13. A method for producing a plant or plant cell, comprising introducinginto a plant cell a gene repair oligonucleobase (GRON) with a targetedmutation in an Acetyl-Coenzyme A carboxylase (ACCase) gene to produce aplant cell with an ACCase gene that expresses an ACCase proteincomprising a mutation at one or more amino acid positions correspondingto a position selected from the group consisting of 1781, 1783, 1786,2078, 2079, 2080 and 2088 based on the numbering of the blackgrassreference sequence SEQ ID NO:1 or at an analogous amino acid residue inan ACCase paralog.14. A method for producing a plant or plant cell, comprising introducinginto a plant cell a DNA cutter and a gene repair oligonucleobase (GRON)with a targeted mutation in an Acetyl-Coenzyme A carboxylase (ACCase)gene to produce a plant cell with an ACCase gene that expresses anACCase protein comprising a mutation at one or more amino acid positionscorresponding to a position selected from the group consisting of 1781,1783, 1786, 2078, 2079, 2080 and 2088 based on the numbering of theblackgrass reference sequence SEQ ID NO:1 or at an analogous amino acidresidue in an ACCase paralog.15. A fertile plant comprising an Acetyl-Coenzyme A carboxylase (ACCase)gene that encodes a protein comprising a mutation at position 2078 basedon the numbering of the blackgrass reference sequence SEQ ID NO:1 or atan analogous amino acid residue in an ACCase paralog.16. A fertile rice plant comprising an Acetyl-Coenzyme A carboxylase(ACCase) gene that encodes a protein comprising a mutation at position2078 based on the numbering of the blackgrass reference sequence SEQ IDNO:1 or at an analogous amino acid residue in an ACCase paralog.17. A plant cell comprising an Acetyl-Coenzyme A carboxylase (ACCase)gene that encodes a protein comprising a mutation at position 2078 basedon the numbering of the blackgrass reference sequence SEQ ID NO:1 or atan analogous amino acid residue in an ACCase paralog and that furthercomprises an Acetyl-Coenzyme A carboxylase (ACCase) gene that encodes aprotein comprising a mutation at one or more amino acid positions, saidpositions selected from the group consisting of 1781, 1783, 1786, 2079,2080 and 2088 based on the numbering of the blackgrass referencesequence SEQ ID NO:1 or at an analogous amino acid residue in an ACCaseparalog.18. A fertile plant comprising an Acetyl-Coenzyme A carboxylase (ACCase)gene that encodes a protein comprising a mutation at position 2078 basedon the numbering of the blackgrass reference sequence SEQ ID NO:1 or atan analogous amino acid residue in an ACCase paralog and that furthercomprises an Acetyl-Coenzyme A carboxylase (ACCase) gene that encodes aprotein comprising a mutation at one or more amino acid positions, saidpositions selected from the group consisting of 1781, 1783, 1786, 2079,2080 and 2088 based on the numbering of the blackgrass referencesequence SEQ ID NO:1 or at an analogous amino acid residue in an ACCaseparalog.19. A method of causing a genetic change in a Acetyl-Coenzyme Acarboxylase (ACCase) gene in a cell, wherein said genetic change causesa change in the Acetyl-Coenzyme A carboxylase (ACCase) protein atposition 2078 based on the numbering of the blackgrass referencesequence SEQ ID NO:1 or at an analogous amino acid residue in an ACCaseparalog said method comprising exposing said cell to a modified GRON.20. A method of causing a genetic change in a Acetyl-Coenzyme Acarboxylase (ACCase) gene in a cell, wherein said genetic change causesa change in the Acetyl-Coenzyme A carboxylase (ACCase) protein atposition 2078 based on the numbering of the blackgrass referencesequence SEQ ID NO:1 or at an analogous amino acid residue in an ACCaseparalog said method comprising exposing said cell to a DNA cutter and amodified GRON.21. The method, plant or cell of any of the preceding embodiments,wherein said mutation or change in an Acetyl-Coenzyme A carboxylase(ACCase) gene, if present results in an Acetyl-Coenzyme A carboxylase(ACCase) protein comprising one or more selected from the groupconsisting of an isoleucine to alanine at a position corresponding toposition 1781 of SEQ ID NO: 1; an isoleucine to leucine at a positioncorresponding to position 1781 of SEQ ID NO: 1; an isoleucine tomethionine at a position corresponding to position 1781 of SEQ ID NO: 1;an isoleucine to asparagine at a position corresponding to position 1781of SEQ ID NO:1; an isoleucine to serine at a position corresponding toposition 1781 of SEQ ID NO: 1; an isoleucine to threonine at a positioncorresponding to position 1781 of SEQ ID NO:1; an isoleucine to valineat a position corresponding to position 1781 of SEQ ID NO: 1; a glycineto cysteine at a position corresponding to position 1783 of SEQ ID NO:1; an alanine to proline at a position corresponding to position 1786 ofSEQ ID NO: 1; an aspartate to glycine at a position corresponding toposition 2078 of SEQ ID NO: 1; an aspartate to lysine at a positioncorresponding to position 2078 of SEQ ID NO: 1; an aspartate tothreonine at a position corresponding to position 2078 of SEQ ID NO: 1;a serine to phenylalanine at a position corresponding to position 2079of SEQ ID NO: 1; a lysine to glutamate at a position corresponding toposition 2080 of SEQ ID NO: 1; a cysteine to phenylalanine at a positioncorresponding to position 2088 of SEQ ID NO: 1; a cysteine to glycine ata position corresponding to position 2088 of SEQ ID NO:1; a cysteine tohistidine at a position corresponding to position 2088 of SEQ ID NO:1; acysteine to lysine at a position corresponding to position 2088 of SEQID NO: 1; a cysteine to leucine at a position corresponding to position2088 of SEQ ID NO: 1; a cysteine to asparagine at a positioncorresponding to position 2088 of SEQ ID NO: 1; a cysteine to proline ata position corresponding to position 2088 of SEQ ID NO:1; a cysteine toglutamine at a position corresponding to position 2088 of SEQ ID NO: 1;a cysteine to arginine at a position corresponding to position 2088 ofSEQ ID NO: 1; a cysteine to serine at a position corresponding toposition 2088 of SEQ ID NO: 1; a cysteine to threonine at a positioncorresponding to position 2088 of SEQ ID NO: 1; a cysteine to valine ata position corresponding to position 2088 of SEQ ID NO: 1; and acysteine to a tryptophan at a position corresponding to position 2088 ofSEQ ID NO: 1.22. The plant or cell of any of the preceding embodiments, or a plant orplant cell made by any of the methods of the preceding embodiments,wherein said plant or cell comprises an Acetyl-Coenzyme A carboxylase(ACCase) protein comprising one or more selected from the groupconsisting of an isoleucine to alanine at a position corresponding toposition 1781 of SEQ ID NO: 1; an isoleucine to leucine at a positioncorresponding to position 1781 of SEQ ID NO: 1; an isoleucine tomethionine at a position corresponding to position 1781 of SEQ ID NO: 1;an isoleucine to asparagine at a position corresponding to position 1781of SEQ ID NO:1; an isoleucine to serine at a position corresponding toposition 1781 of SEQ ID NO: 1; an isoleucine to threonine at a positioncorresponding to position 1781 of SEQ ID NO: 1; an isoleucine to valineat a position corresponding to position 1781 of SEQ ID NO:1; a glycineto cysteine at a position corresponding to position 1783 of SEQ ID NO:1; an alanine to proline at a position corresponding to position 1786 ofSEQ ID NO: 1; an aspartate to glycine at a position corresponding toposition 2078 of SEQ ID NO: 1; an aspartate to lysine at a positioncorresponding to position 2078 of SEQ ID NO:1; an aspartate to threonineat a position corresponding to position 2078 of SEQ ID NO: 1; a serineto phenylalanine at a position corresponding to position 2079 of SEQ IDNO: 1; a lysine to glutamate at a position corresponding to position2080 of SEQ ID NO: 1; a cysteine to phenylalanine at a positioncorresponding to position 2088 of SEQ ID NO: 1; a cysteine to glycine ata position corresponding to position 2088 of SEQ ID NO: 1; a cysteine tohistidine at a position corresponding to position 2088 of SEQ ID NO: 1;a cysteine to lysine at a position corresponding to position 2088 of SEQID NO: 1; a cysteine to leucine at a position corresponding to position2088 of SEQ ID NO: 1; a cysteine to asparagine at a positioncorresponding to position 2088 of SEQ ID NO: 1; a cysteine to proline ata position corresponding to position 2088 of SEQ ID NO: 1; a cysteine toglutamine at a position corresponding to position 2088 of SEQ ID NO:1; acysteine to arginine at a position corresponding to position 2088 of SEQID NO:1; a cysteine to serine at a position corresponding to position2088 of SEQ ID NO:1; a cysteine to threonine at a position correspondingto position 2088 of SEQ ID NO:1; a cysteine to valine at a positioncorresponding to position 2088 of SEQ ID NO: 1; and a cysteine to atryptophan at a position corresponding to position 2088 of SEQ ID NO: 1.23. The plant or cell of any of the preceding embodiments, or a plant orcell made by any of the methods of the preceding embodiments, whereinsaid plant or plant cell comprises an Acetyl-Coenzyme A carboxylase(ACCase) gene that encodes a protein comprising a mutation at one ormore amino acid positions, said positions selected from the groupconsisting of 1781, 1783, 1786, 2078, 2079, 2080 and 2088 based on thenumbering of the blackgrass reference sequence SEQ ID NO:1 or at ananalogous amino acid residue in an ACCase paralog.24. The plant or cell of any of the preceding embodiments, or a plant orcell made by any of the methods of the preceding embodiments, whereinsaid plant or cell comprises an Acetyl-Coenzyme A carboxylase (ACCase)gene that encodes a protein comprising a mutation at position 2078 basedon the numbering of the blackgrass reference sequence SEQ ID NO:1 or atan analogous amino acid residue in an ACCase paralog and that furthercomprises an Acetyl-Coenzyme A carboxylase (ACCase) gene that encodes aprotein comprising a mutation at one or more amino acid positions, saidpositions selected from the group consisting of 1781, 1783, 1786, 2079,2080 and 2088 based on the numbering of the blackgrass referencesequence SEQ ID NO:1 or at an analogous amino acid residue in an ACCaseparalog.

In each of the foregoing ACCase embodiments 11-24, whether methods,plants, cells, or otherwise, the following are suitable mutations foruse therein:

Amino Amino Acid Codon Acid Codon Change Change Change Change I1781AATA > GCT C2088F TGC > TTT ATA > GCC TGC > TTC ATA > GCA C2088G TGC >GGT ATA > GCG TGC > GGC I1781L ATA > CTT TGC > GGA ATA > CTC TGC > GGGATA > CTA C2088H TGC > CAT ATA > CTG TGC > CAC ATA > TTA C2088K TGC >AAA ATA > TTG TGC > AAG I1781M ATA > ATG C2088L TGC > CTT I1781N ATA >AAT TGC > CTC ATA > AAC TGC > CTA I1781S ATA > TCT TGC > CTG ATA > TCCTGC > TTA ATA > TCA TGC > TTG ATA > TCG C2088N TGC > AAT I1781T ATA >ACT TGC > AAC ATA > ACC C2088P TGC > CCT ATA > ACA TGC > CCC ATA > ACGTGC > CCA I1781V ATA > GTT TGC > CCG ATA > GTC C2088Q TGC > CAA ATA >GTA TGC > CAG ATA > GTG C2088R TGC > CGT G1783C GGA > TGT TGC > CGCGGA > TGC TGC > CGA A1786P GCT > CCT TGC > CGG GCT > CCC TGC > AGA GCT >CCA TGC > AGG GCT > CCG C2088S TGC > TCT D2078G GAT > GGT TGC > TCCGAT > GGC TGC > TCA GAT > GGA TGC > TCG GAT > GGG C2088T TGC > ACTD2078K GAT > AAA TGC > ACC GAT > AAG TGC > ACA D2078T GAT > ACT TGC >ACG GAT > ACC C2088V TGC > GTT GAT > ACA TGC > GTC GAT > ACG TGC > GTAS2079F AGC > TTT TGC > GTG AGC > TTC C2088W TGC > TGG K2080E AAG > GAAAAG > GAG

Alternative mutations include, but are not limited to, the following:

S2079A AGC > GCT AGC > GCC AGC > GCA AGC > GCG G1783A GGA > GCT GGA >GCC GGA > GCA GGA > GCG A1786G GCT > GGT GCT > GGC GCT > GGA GCT > GGG

With regard to embodiments 11-24, corresponding positions to 1781 m1783, 1786, 2078, 2079, and 2080 based on the numbering of theblackgrass reference sequence are well known in the art and readilyobtainable from appropriate sequence databases. By way of example, thefollowing table shows the corresponding positions in the rice ACCasesequence:

Am OsI OsJ I1781 I1792 I1779 G1783 G1794 G1781 A1786 A1797 A1784 D2078D2089 D2076 S2079 S2090 S2077 K2080 K2091 K2078 C2088 C2099 C2086 Am:Alopecurus myosuroide; OsI: Oryza sativa indica variety; OsJ: Oryzasativa japonica variety25. A method for producing a plant or plant cell with a mutated EPSPSgene, comprising introducing into a plant cell a gene repairoligonucleobase (GRON) with a targeted mutation in an 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene to produce a plantcell with an EPSPS gene that expresses an EPSPS protein comprising amutation at one or more amino acid positions corresponding to a positionselected from the group consisting of 96, 97 and 101 based on thenumbering of the amino acid sequence for the Escherichia coli referencesequence SEQ ID NO:2 or at an analogous amino acid residue in an EPSPSparalog.26. A method for producing a plant or plant cell with a mutated EPSPSgene, comprising introducing into a plant cell a DNA cutter and a generepair oligonucleobase (GRON) with a targeted mutation in an 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene to produce a plantcell with an EPSPS gene that expresses an EPSPS protein comprising amutation at one or more amino acid positions corresponding to a positionselected from the group consisting of 96, 97 and 101 based on thenumbering of the amino acid sequence for the Escherichia coli referencesequence SEQ ID NO:2 or at an analogous amino acid residue in an EPSPSparalog.27. A plant or cell with a mutated EPSPS gene, wherein said plant orcell is made by a method introducing into a plant cell a DNA cutter anda gene repair oligonucleobase (GRON) with a targeted mutation in an5-enol pyruvylshikimate-3-phosphate synthase (EPSPS) gene to produce aplant cell with an EPSPS gene that expresses an EPSPS protein comprisinga mutation at one or more amino acid positions corresponding to aposition selected from the group consisting of 96, 97 and 101 based onthe numbering of the amino acid sequence for the Escherichia colireference sequence SEQ ID NO:2 or at an analogous amino acid residue inan EPSPS paralog.28. The plant or cell of any of the preceding embodiments, or a plant orcell made by any of the methods of the preceding embodiments, whereinthe plant or plant cell expresses an EPSPS protein comprising a mutationat one or more amino acid positions are selected from the groupconsisting of a glycine to alanine at a position corresponding toposition 96 of SEQ ID NO:2; a threonine to isoleucine at a positioncorresponding to position 97 of SEQ ID NO:2; a proline to alanine at aposition corresponding to position 101 of SEQ ID NO:2; a proline toserine at a position corresponding to position 101 of SEQ ID NO:2; and aproline to threonine at a position corresponding to position 101 of SEQID NO:2.29. The plant or cell of any of the preceding embodiments, or a plant orcell made by any of the methods of the preceding embodiments, whereinthe plant or plant cell expresses an EPSPS protein comprising mutationcombinations selected from the group consisting of a threonine toisoleucine at a position corresponding to position 97 of SEQ ID NO:2 anda proline to alanine at a position corresponding to position 101 of SEQID NO:2; a threonine to isoleucine at a position corresponding toposition 97 of SEQ ID NO:2 and a proline to alanine at a positioncorresponding to position 101 of SEQ ID NO:2; a threonine to isoleucineat a position corresponding to position 97 of SEQ ID NO:2 and a prolineto serine at a position corresponding to position 101 of SEQ ID NO:2;and a threonine to isoleucine at a position corresponding to position 97of SEQ ID NO:2 and a proline to threonine at a position corresponding toposition 101 of SEQ ID NO:2.

With regard to embodiments 25-30, corresponding positions to 96, 97 and101 based on the numbering of the Escherichia coli reference sequenceSEQ ID NO:2 are well known in the art and readily obtainable fromappropriate sequence databases. See e.g., U.S. Pat. No. 8,268,622. Byway of example, the following table shows the corresponding positions inthe flax EPSPS sequence:

Flax EPSPS E. coli EPSPS Gene1 Gene2 G96 G176 G177 T97 T177 T178 P101P181 P182 E. coli EPSPS sequence is AroA having the sequenceMESLTLQPIARVDGTINLPGSKTVSNRALLLAALAHGKTVLTNLLDSDDVRHMLNALTALGVSYTLSADRTRCEIIGNGGPLHAEGALELFLGNAGTAMRPLAAALCLGSNDIVLTGEPRMKERPIGHLVDALRLGGAKITYLEQENYPPLRLQGGFTGGNVDVDGSVSSQFLTALLMTAPLAPEDTVIRIKGDLVSKPYIDITLNLMKTFGVEIENQHYQQFVVKGGQSYQSPGTYLVEGDASSASYFLAAAAIKGGTVKVTGIGRNSMQGDIRFADVLEKMGATICWGDDYISCTRGELNAIDMDMNHIPDAAMTIATAALFAKGTTRLRNIYNWRVKETDRLFAMATELRKVGAEVEEGHDYIRITPPEKLNFAEIATYNDHRMAMCFSLVALSDTPVTILDPKCTAKTFPDYFEQLARISQAA (SEQ ID NO: 10) Flax gene 1 sequence islcl - g41452_1333 having the sequenceMALVTKICGGANAVALPATFGTRRTKSISSSVSFRSSTSPPSLKQRRRSGNVAAAAAAPLRVSASLTTAAEKASTVPEEVVLQPIKDISGIVTLPGSKSLSNRILLLAALSEGTTVVDNLLNSDDVHYMLGALKTLGLNVEHSSEQKRAIVEGCGGVFPVGKLAKNDIELFLGNAGTAMRPLTAAVTAAGGNSSYILDGVPRMRERPIGDLVVGLKQLGADVTCSSTSCPPVHVNGQGGLPGGKVKLSGSISSQYLTALLMAAPLALGDVEIEIVDKLISVPYVDMTLKLMERFGVAVEHSGSWDRFFVKGGQKYKSPGNAYVEGDASSASYFLAGAAITGGTITVEGCGTSSLQGDVKFAEVLEKMGAKVIWTENSVTVTGPPRDASGRKHLRAVDVNMNKMPDVAMTLAVVALYADGPTAIRDVASWRVKETERMIAICTELRKLGATVEEGPDYCIITPPEKLNIAEIDTYDDHRMAMAFSLAACADVPVTIRDPGC TKKTFPDYFEVLERYTKH(SEQ ID NO: 11) Flax gene 2 sequence is lcl - g40547_1271 having thesequence MAQVTKICGGANAVALPATFGTRRTKSISSSVSFRSSTSPPSLKQRRLLGNVAAAAAAAPLRISASLATAAEKASTVPEEIVLQPIKDISGIVTLPGSKSLSNRILLLAALSEGKTVVDNLLNSDDVHYMLGALKTLGLNVEHSSEQKRAIVEGRGGVFPVGKLGKNDIELFLGNAGTAMRPLTAAVTAAGGNSSYILDGVPRMRERPIGDLVVGLKQLGADVSCSSTSCPPVHVNAKGGLPGGKVKLSGSISSQYLTALLMAAPLALGDVEIEIVDKLISVPYVDMTLKLMERFGVAVEHSGSWDRFFVKGGQKYKSPGNAYVEGDASSASYFLAGAAITGGTITVEGCGTSSLQGDVKFAEVLEKMGAKVTWTETSVTVTGPPRDASGKKHLRAVDVNMNKMPDVAMTLAVVALYADGPTAIRDVASWRVKETERMIAVCTELRKLGATVEEGPDYCIITPPEKLSIAEIDTYDDHRMAMAFSLAACADVPVTIRDPG CTKKTFPDYFEVLERYTKH(SEQ ID NO: 12)30. The method or cell of any of the preceding embodiments, wherein saidDNA cutter is one or more selected from a CRISPR, a TALEN, a zincfinger, meganuclease, and a DNA-cutting antibiotic.31. The method or cell of any of the preceding embodiments, wherein saidDNA cutter is a CRISPR or a TALEN.32. The method or cell of any of the preceding embodiments, wherein saidDNA cutter is a CRISPR.33. The method or cell of any of the preceding embodiments, wherein saidDNA cutter is a TALEN.34. The method or cell of any of the preceding embodiments, wherein saidDNA cutter is one or more DNA-cutting antibiotics selected from thegroup consisting of bleomycin, zeocin, phleomycin, tallysomycin andpepleomycin.35. The method or cell of any of the preceding embodiments, wherein saidDNA cutter is zeocin.36. The method or cell of any of the preceding embodiments, wherein saidGRON is single stranded.37. The method or cell of any of the preceding embodiments, wherein theGRON is a chemically protected oligonucleotide.38. The method or cell of any of the preceding embodiments, wherein theGRON comprises a chemically protected oligonucleotide protected at the5′ end.39. The method or cell of any of the preceding embodiments, wherein theGRON comprises a chemically protected oligonucleotide protected at the3′ end.40. The method or cell of any of the preceding embodiments, wherein theGRON comprises a chemically protected oligonucleotide protected at the5′ and 3′ ends.41. The method or cell of any of the preceding embodiments, wherein theGRON comprises one or more selected from a Cy3 group, a 3PS group, idCgroup, and a 2′-O-methyl group.42. The method or cell of any of the preceding embodiments, wherein theGRON comprises a Cy3 group.43. The method or cell of any of the preceding embodiments, wherein theGRON comprises two or more Cy3 groups.44. The method or cell of any of the preceding embodiments, wherein theGRON comprises a Cy3 group at the first (ultimate) base on the 5′ end.45. The method or cell of any of the preceding embodiments, wherein theGRON comprises an idC group at the first (ultimate) base on the 5′ end.46. The method or cell of any of the preceding embodiments, wherein theGRON comprises a Cy3 group at the first (ultimate) base on the 3′ end.47. The method or cell of any of the preceding embodiments, wherein theGRON comprises an idC group at the first (ultimate) base on the 3′ end.48. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 3PS group.49. The method or cell of any of the preceding embodiments, wherein theGRON comprises two or more 3PS groups.50. The method or cell of any of the preceding embodiments, wherein theGRON comprises three or more 3PS groups.51. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 3PS group at the first (ultimate) base on the 5′ end.52. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 3PS group at the second (penultimate) base on the 5′end.53. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 3PS group at the third (antepenultimate) base on the 5′end.54. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 3PS group at the first (ultimate) base on the 3′ end.55. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 3PS group at the second to last (penultimate) base onthe 3′ end.56. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 3PS group at the third to last (antepenultimate) baseon the 3′ end.57. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 3PS group at the first (ultimate) bases on both the 5′and 3′ end.58. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 3PS group at the first two bases on both the 5′ and the3′ end.59. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 3PS group at the first three bases on both the 5′ andthe 3′ end.60. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 2′-O-methyl group.61. The method or cell of any of the preceding embodiments, wherein theGRON comprises two or more 2′-O-methyl groups.62. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 2′-O-methyl group at the first (ultimate) base on the5′ end.63. The method or cell of any of the preceding embodiments, wherein theGRON has a 2′-O-methyl group at the first base on the 5′ end and doesnot have any other 2′-O-methyl groups.64. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 2′-O-methyl group on each of the first two or morebases at the 5′ end.65. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 2′-O-methyl group on each of the first three or morebases at the 5′ end.66. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 2′-O-methyl group on each of the first four or morebases at the 5′ end.67. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 2′-O-methyl group on each of the first five or morebases at the 5′ end.68. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 2′-O-methyl group on each of the first six or morebases at the 5′ end.69. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 2′-O-methyl group on each of the first seven or morebases at the 5′ end.70. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 2′-O-methyl group on each of the first eight or morebases at the 5′ end.71. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 2′-O-methyl group on each of the first nine or morebases at the 5′ end.72. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 2′-O-methyl group on each of the first ten or morebases at the 5′ end.73. The method or cell of any of the preceding embodiments, wherein theGRON comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 RNA bases at the 5′ end.74. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 2′-O-methyl group at the first (ultimate) base on the3′ end.75. The method or cell of any of the preceding embodiments, wherein theGRON has a 2′-O-methyl group at the first base on the 3′ end and doesnot have any other 2′-O-methyl groups.76. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 2′-O-methyl group on each of the first two (ultimateand penultimate) or more bases at the 3′ end.77. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 2′-O-methyl group on each of the first three (ultimateand penultimate, and antepenultimate) or more bases at the 3′ end.78. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 2′-O-methyl group on each of the first four or morebases at the 3′ end.79. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 2′-O-methyl group on each of the first five or morebases at the 3′ end.80. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 2′-O-methyl group on each of the first six or morebases at the 3′ end.81. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 2′-O-methyl group on each of the first seven or morebases at the 3′ end.82. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 2′-O-methyl group on each of the first eight or morebases at the 3′ end.83. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 2′-O-methyl group on each of the first nine or morebases at the 3′ end.84. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 2′-O-methyl group on each of the first ten or morebases at the 3′ end.85. The method or cell of any of the preceding embodiments, wherein theGRON comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 RNA bases at the 3′ end.86. The method or cell of any of the preceding embodiments, wherein saidGRON has a wobble base pair relative to the target sequence for thegenetic change.87. The method or cell of any of the preceding embodiments, wherein saidGRON is between 15 and 60 nucleotides in length.88. The method or cell of any of the preceding embodiments, wherein saidGRON is 41 nucleotides in length.89. The method or cell of any of the preceding embodiments, wherein saidGRON is between 50 and 110 nucleotides in length.90. The method or cell of any of the preceding embodiments, wherein saidGRON is 101 nucleotides in length.91. The method or cell of any of the preceding embodiments, wherein saidGRON is between 150 and 210 nucleotides in length.92. The method or cell of any of the preceding embodiments, wherein saidGRON is 201 nucleotides in length.93. The method or cell of any of the preceding embodiments, wherein saidGRON is between 70 and 210 nucleotides in length.94. The method or cell of any of the preceding emodiments, wherein saidGRON is longer than 70 nucleotides in length.95. The method or cell of any of the preceding emodiments, wherein saidGRON is longer than 100 nucleotides in length.96. The method or cell of any of the preceding embodiments, wherein saidGRON is longer than 165 nucleotides in length.97. The method or cell of any of the preceding embodiments, wherein saidGRON is longer than 175 nucleotides in length.98. The method or cell of any of the preceding embodiments, wherein saidGRON is longer than 185 nucleotides in length.99. The method or cell of any of the preceding embodiments, wherein saidGRON is longer than 195 nucleotides in length.100. The method or cell of any of the preceding embodiments, whereinsaid GRON is longer than 200 nucleotides in length.101. The method or cell of any of the preceding embodiments, whereinsaid GRON is longer than 210 nucleotides in length.102. The method or cell of any of the preceding embodiments, whereinsaid GRON is longer than 220 nucleotides in length.103. The method or cell of any of the preceding embodiments, whereinsaid GRON is longer than 230 nucleotides in length.104. The method or cell of any of the preceding embodiments, whereinsaid GRON is longer than 240 nucleotides in length.105. The method or cell of any of the preceding embodiments, whereinsaid GRON is longer than 250 nucleotides in length.106. The method or cell of any of the preceding embodiments, whereinsaid GRON is longer than 260 nucleotides in length.107. The method or cell of any of the preceding embodiments, whereinsaid GRON is longer than 270 nucleotides in length.108. The method or cell of any of the preceding embodiments, whereinsaid GRON is longer than 280 nucleotides in length.109. The method or cell of any of the preceding embodiments, whereinsaid GRON is longer than 290 nucleotides in length.110. The method or cell of any of the preceding embodiments, whereinsaid GRON is longer than 300 nucleotides in length.111. The method or cell of any of the preceding embodiments, whereinsaid GRON is longer than 400 nucleotides in length.112. The method or cell of any of the preceding embodiments, whereinsaid GRON is longer than 500 nucleotides in length.113. The method or cell of any of the preceding embodiments, whereinsaid GRON is longer than 600 nucleotides in length.114. The method or cell of any of the preceding embodiments, whereinsaid GRON is longer than 700 nucleotides in length.115. The method or cell of any of the preceding embodiments, whereinsaid GRON is longer than 800 nucleotides in length.116. The method or cell of any of the preceding embodiments, whereinsaid GRON is longer than 900 nucleotides in length.117. The method or cell of any of the preceding embodiments, whereinsaid GRON is longer than 1000 nucleotides in length.118. The method or cell of any of the preceding embodiments wherein saidplant is selected from the group consisting of canola, sunflower, corn,tobacco, sugar beet, cotton, maize, wheat, barley, rice, alfalfa,barley, sorghum, tomato, mango, peach, apple, pear, strawberry, banana,melon, cassava, potato, carrot, lettuce, onion, soy bean, soya spp,sugar cane, pea, chickpea, field pea, fava bean, lentils, turnip,rutabaga, brussel sprouts, lupin, cauliflower, kale, field beans,poplar, pine, eucalyptus, grape, citrus, triticale, alfalfa, rye, oats,turf and forage grasses, flax, oilseed rape, mustard, cucumber, morningglory, balsam, pepper, eggplant, marigold, lotus, cabbage, daisy,carnation, tulip, iris, and lily.119. The method or cell of any of the preceding embodiments wherein saidplant is canola.120. The method or cell of any of the preceding embodiments wherein saidplant is corn 121. The method or cell of any of the precedingembodiments wherein said plant is maize.122. The method or cell of any of the preceding embodiments wherein saidplant is rice.123. The method or cell of any of the preceding embodiments wherein saidplant is sorghum.124. The method or cell of any of the preceding embodiments wherein saidplant is potato.125. The method or cell of any of the preceding embodiments wherein saidplant is soy bean.126. The method or cell of any of the preceding embodiments wherein saidplant is flax.127. The method or cell of any of the preceding embodiments wherein saidplant is oilseed rape.128. The method or cell of any of the preceding embodiments wherein saidplant is cassava.129. The method or cell of any of the preceding embodiments wherein saidplant is sunflower.130. A method of causing a genetic change in a plant cell, said methodcomprising exposing said cell to a CRISPR and a modified GRON.131. The method or cell of any of the preceding embodiments whereinmultiple genetic changes are made.132. The method or cell of any of the preceding embodiments wherein twoor more guide RNAs are used.133. The method or cell of any of the preceding embodiments wherein eachof the more than one guide RNAs is complimentary to a different targetfor genetic change.134. The method or cell of any of the preceding embodiments wherein theCRISPR includes a nickase.135. The method or cell of any of the preceding embodiments wherein theDNA cutter includes two or more nickases.136. The method or cell of any of the preceding embodiments wherein twoor more nickases cuts on opposite strands of the target nucleic acidsequence.137. The method or cell of any of the preceding embodiments wherein twoor more nickases cuts on the same strand of the target nucleic acidsequence.138. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 2′-O-methyl group at the first (ultimate) base on the3′ end.139. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 2′-O-methyl group at the first (ultimate) base on the3′ end and does not have any other 2′-O-methyl groups.140. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 2′-O-methyl group on each of the first two or morebases at the 3′ end.141. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 2′-O-methyl group on each of the first three or morebases at the 3′ end.142. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 2′-O-methyl group on each of the first four or morebases at the 3′ end.143. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 2′-O-methyl group on each of the first five or morebases at the 3′ end.144. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 2′-O-methyl group on each of the first six or morebases at the 3′ end.145. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 2′-O-methyl group on each of the first seven or morebases at the 3′ end.146. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 2′-O-methyl group on each of the first eight or morebases at the 3′ end.147. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 2′-O-methyl group on each of the first nine or morebases at the 3′ end.148. The method or cell of any of the preceding embodiments, wherein theGRON comprises a 2′-O-methyl group on each of the first ten or morebases at the 3′ end.149. The method or cell of any of the preceding embodiments, wherein theGRON comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 RNA bases at the 3′ end.150. The method or cell of any of the preceding embodiments, wherein theGRON does not comprise a 2′-O-methyl group at the first (ultimate) baseon the 3′ end.151. The method or cell of any of the preceding embodiments, wherein theGRON does not comprise a 2′-O-methyl group on any of the first two ormore bases at the 3′ end.152. The method or cell of any of the preceding embodiments, wherein theGRON does not comprise a 2′-O-methyl group on any of the first three ormore bases at the 3′ end.153. The method or cell of any of the preceding embodiments, wherein theGRON does not comprise a 2′-O-methyl group on any of the first four ormore bases at the 3′ end.154. The method or cell of any of the preceding embodiments, wherein theGRON does not comprise a 2′-O-methyl group on any of the first five ormore bases at the 3′ end.155. The method or cell of any of the preceding embodiments, wherein theGRON does not comprise a 2′-O-methyl group on any of the first six ormore bases at the 3′ end.156. The method or cell of any of the preceding embodiments, wherein theGRON does not comprise a 2′-O-methyl group on any of the first seven ormore bases at the 3′ end.157. The method or cell of any of the preceding embodiments, wherein theGRON does not comprise a 2′-O-methyl group on any of the first eight ormore bases at the 3′ end.158. The method or cell of any of the preceding embodiments, wherein theGRON does not comprise a 2′-O-methyl group on any of the first nine ormore bases at the 3′ end.159. The method or cell of any of the preceding embodiments, wherein theGRON does not comprise a 2′-O-methyl group on any of the first ten ormore bases at the 3′ end.160. The method or cell of any of the preceding embodiments, wherein theGRON does not comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 RNA bases at the3′ end.161. A non-transgenic herbicide resistant or tolerant plant made by themethod or from the cell of one any of the preceding embodiments.162. The method or cell or plant of any of the preceding embodiments,wherein said plant cell has a genetic change or mutation inAcetyl-Coenzyme A carboxylase (ACCase) and is selected from the groupconsisting of barley, maize, millet, oats, rye, rice, sorghum,sugarcane, turf grasses, and wheat.163. The method or cell or plant of any of the preceding embodiments,wherein said plant cell has a genetic change or mutation inAcetyl-Coenzyme A carboxylase (ACCase) and is resistant or tolerant toone or more herbicides.164. The method or cell or plant of any of the preceding embodiments,wherein said plant cell has a genetic change or mutation inAcetyl-Coenzyme A carboxylase (ACCase), is resistant to one or moreACCase-inhibiting herbicides.165. The method or cell or plant of any of the preceding embodiments,wherein said plant cell has a genetic change or mutation inAcetyl-Coenzyme A carboxylase (ACCase), is resistant to one or moreherbicides selected from the group consisting of alloxydim, butroxydim,clethodim, cloproxydim, cycloxydim, sethoxydim, tepraloxydim,tralkoxydim, chlorazifop, clodinafop, clofop, diclofop, fenoxaprop,fenoxaprop-P, fenthiaprop, fluazifop, fluazifop-P, haloxyfop,haloxyfop-P, isoxapyrifop, propaquizafop, quizalofop, quizalofop-P,trifop, pinoxaden, agronomically acceptable salts and esters of any ofthese herbicides, and combinations thereof.166. The method or cell or plant of any of the preceding embodiments,wherein said plant cell has a genetic change or mutation in5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), and wherein saidplant cell is selected from the group consisting of corn, wheat, rice,barley, sorghum, oats, rye, sugarcane, soybean, cotton, sugarbeet,oilseed rape, canola, flax, cassava, sunflower, potato, tobacco, tomato,alfalfa, poplar, pine, eucalyptus, apple, lettuce, peas, lentils, grapeand turf grasses.167. The method or cell or plant of any of the preceding embodiments,wherein said plant or plant cell has a genetic change or mutation in5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), and wherein plantor plant cell is resistant to at least one herbicide.168. The method or cell or plant of any of the preceding embodiments,wherein said plant or plant cell has a genetic change or mutation in5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), and wherein plantor plant cell is resistant to a herbicide of the phosphonomethylglycinefamily.169. The method or cell or plant of any of the preceding embodiments,wherein said plant or plant cell has a genetic change or mutation in5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), and wherein plantor plant cell is resistant to glyphosate.170. The method or cell or plant of any of the preceding embodiments,wherein said plant or plant cell has a genetic change or mutation in5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), and wherein plantor plant cell is selected from the group consisting of corn, wheat,rice, barley, sorghum, oats, rye, sugarcane, soybean, cotton, sugarbeet,oilseed rape, canola, flax, cassava, sunflower, potato, tobacco, tomato,alfalfa, poplar, pine, eucalyptus, apple, lettuce, peas, lentils, grapeand turf grasses.171. The method or cell or plant of any of the preceding embodiments,wherein the genetic change or mutation in the cell occurs at one alleleof the gene.172. The method or cell or plant of any of the preceding embodiments,wherein the genetic change or mutation in the cell occurs at two allelesof the gene.173. The method or cell or plant of any of the preceding embodiments,wherein the genetic change or mutation in the cell occurs at threealleles of the gene.174. The method or cell or plant of any of the preceding embodiments,wherein the genetic change or mutation in the cell occurs at fouralleles of the gene.175. The method or cell or plant of any of the preceding embodiments,wherein the genetic change or mutation in the cell occurs at one, two,three, four, five, six, seven, eight, nine, ten, eleven, or twelvealleles of the gene.176. The method or cell or plant of any of the preceding embodiments,wherein the genetic change or mutation in the cell comprises a deletionor insertion resulting in a knockout of one allele of the gene.177. The method or cell or plant of any of the preceding embodiments,wherein the genetic change or mutation in the cell comprises a deletionor insertion resulting in a knockout of two alleles of the gene.178. The method or cell or plant of any of the preceding embodiments,wherein the genetic change or mutation in the cell comprises a deletionor insertion resulting in a knockout of three alleles of the gene.179. The method or cell or plant of any of the preceding embodiments,wherein the genetic change or mutation in the cell comprises a deletionor insertion resulting in a knockout of four alleles of the gene.180. The method or cell or plant of any of the preceding embodiments,wherein the genetic change or mutation in the cell comprises a deletionor insertion resulting in a knockout of one, two, three, four, five,six, seven, eight, nine, ten, eleven, or twelve alleles of the gene.181. The method or cell or plant of any of the preceding embodiments,wherein the genetic change or mutation in the cell occurs at one alleleof the gene and a second allele of the gene comprises a deletion orinsertion resulting in a knockout of said second allele.182. The method or cell or plant of any of the preceding embodiments,wherein the genetic change or mutation in the cell occurs at one alleleof the gene and a second allele and third allele of the gene comprises adeletion or insertion resulting in a knockout of said second allele andsaid third allele.183. The method or cell or plant of any of the preceding embodiments,wherein the genetic change or mutation in the cell occurs at one alleleof the gene and a second allele, third allele, and fourth allele of thegene comprises a deletion or insertion resulting in a knockout of saidsecond allele, said third allele and said fourth allele.184. The method or cell or plant of any of the preceding embodiments,wherein the genetic change in the cell comprises at least one mutationat one allele and at least one knockout in another allele.185. The method or cell or plant of any of the preceding embodiments,wherein the genetic change in the cell comprises at least one mutationat one allele and at least one knockout in at least one other allele.186. The method or cell or plant of any of the preceding embodiments,wherein the genetic change in the cell comprises at least one mutationat one allele and at least one knockout in at least two other alleles.187. The method or cell or plant of any of the preceding embodiments,wherein the genetic change in the cell comprises at least one mutationat one allele and at least one knockout in at least three other alleles.188. The method or cell or plant of any of the preceding embodiments,wherein the genetic change in the cell comprises at least one mutationat one allele and a knockout in all other alleles.

EXAMPLES

The following are examples, which illustrate procedures for practicingthe methods and compositions described herein. These examples should notbe construed as limiting.

Example 1: GRON Length

Sommer et al., (Mol Biotechnol. 33:115-22, 2006) describes a reportersystem for the detection of in vivo gene conversion that relies upon asingle nucleotide change to convert between blue and green fluorescencein green fluorescent protein (GFP) variants. This reporter system wasadapted for use in the following experiments using Arabidopsis thalianaas a model species in order to assess efficiency of GRON mediatedconversion.

In short, for this and the subsequent examples an Arabidopsis thalianaline with multiple copies of a blue fluorescent protein gene was createdby methods known to those skilled in the art (see, e.g., Clough andBrent, 1998). Root-derived meristematic tissue cultures were establishedwith this line, which was used for protoplast isolation and culture(see, e.g., Mathur et al., 1995). GRON delivery into protoplasts wasachieved through polyethylene glycol (PEG) mediated GRON uptake intoprotoplasts. A method consisting of a 96-well dish format, similar tothat described by Fujiwara and Kato (2007) was used. In the followingthe protocol is briefly described. The volumes given are those appliedto individual wells of a 96-well dish.

-   1. Mix 6.25 μl of GRON (80 μM) with 25 μl of Arabidopsis thaliana    BFP transgenic root meristematic tissue-derived protoplasts at 5×10⁶    cells/ml in each well of a 96 well plate.-   2. 31.25 μl of a 40% PEG solution was added and the protoplasts were    mixed.-   3. Treated cells were incubated on ice for 30 min.-   4. To each well 200 μl of W5 solution was added and the cells mixed.-   5. The plates were allowed to incubate on ice for 30 min allowing    the protoplasts to settle to the bottom of each well.-   6. 200 μl of the medium above the settled protoplasts was removed.-   7. 85 μl of culture medium (MSAP, see Mathur et al., 1995) was    added.-   8. The plates were incubated at room temperate in the dark for 48    hours. The final concentration of GRON after adding culture medium    is 8 PM.

In general, samples were analyzed by flow cytometry 48 h after GRONdelivery in order to detect protoplasts whose green and yellowfluorescence is different from that of control protoplasts that aretreated with non-targeting GRONs that does not change the BFP target DNAIn samples treated with targeting GRONs, there is a single C to Tnucleotide difference (coding strand) or G to A nucleotide difference(non-coding strand) that when introduced into the BFP gene by geneediting, results in the synthesis of GFP.

Table 1 shows the sequences of exemplary 101-mer and 201-mer BFP4/NC5′-3PS/3′-3PS GRONs designed for the conversion of a blue fluorescentprotein (BFP) gene to green fluorescence. “3PS” denotes 3 phosphothioatelinkages at each of the 5′ and 3′ oligo ends.

TABLE 1 Exemplary GRON Nucleotide Sequences for BFP to GFP conversionGRON Name GRON Nucleotide Sequence BFP4/NCG* T*C*G TGC TGC TTC ATG TGG TCG GGG TAG CGG CTG AAG CAC 101-merTGC ACG CCG TAG GTG AAG GTG GTC ACG AGG GTG GGC CAGGGC ACG GGC AGC TTG CCG G*T*G* G (SEQ ID NO: 13) BFPO/NCG* T*C*G TGC TGC TTC ATG TGG TCG GGG TAG CGG CTG AAG CAC 101-merTGC ACG CCG TGG GTG AAG GTG GTC ACG AGG GTG GGC CAGGGC ACG GGC AGC TTG CCG G*T*G *G (SEQ ID NO: 14) BFP4/CC *C*A*C CGG CAA GCT GCC CGT GCC CTG GCC CAC CCT CGT GAC 101-merCAC CTT CAC CTA CGG CGT GCA GTG CTT CAG CCG CTA CCC CGACCA CAT GAA GCA GCA C*G*A *C (SEQ ID NO: 15) BFP0/CC*C*A*CCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCAC 101-merCTTCACCCACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCAC*G*A* C (SEQ ID NO: 16) BFP4/NCA*A*G*ATGGTGCGCTCCTGGACGTAGCCTTCGGGCATGGCGGACT 201-merTGAAGAAGTCGTGCTGCTTCATGTGGTCTGGGTAGCGGCTGAAGCACTGCACGCCGTAGGTGAAGGTGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGATGAACTTCAGGGTCAGCTTGCCG TAGGTGGCATCGCCCTCG *C*C*C (SEQ ID NO: 17) BFP0/NCA*A*G*TGGTGCGCTCCTGGACGTAGCCTTCGGGCATGGCGGACTT 201-merGAAGAAGTCGTGCTGCTTCATGTGGTCGGGGTAGCGGCTGAAGCACTGCACGCCGTGGGTGAAGGTGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGATGAACTTCAGGGTCAGCTTGCCG TAGGTGGCATCGCCCTCG *C*C*C (SEQ ID NO: 18) BFP4/CG*G*G*CGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTC 201-merATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGA GCGCACCAT *C*T*T (SEQ lD NO: 19) BFP0/CG*G*G*CGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTC 201-merATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCACCCACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGA GCGCACCAT*C*T*T (SEQ ID NO: 20) *= PS linkage (phosphothioate)

Example 2: Conversion Rates Using 5′Cy3/3′idC Labeled GRONs

The purpose of this series of experiments is to compare the efficienciesof phosphothioate (PS) labeled GRONs (having 3 PS moieties at each endof the GRON) to the 5′Cy3/3′idC labeled GRONs. The 5′Cy3/3′idC labeledGRONs have a 5′ Cy3 fluorophore (amidite) and a 3′ idC reverse base.Efficiency was assessed using conversion of blue fluorescent protein(BFP) to green fluorescence protein (GFP).

In all three experiments, done either by PEG delivery of GRONs intoprotoplasts in individual Falcon tubes (labeled “Tubes”) or in 96-wellplates (labeled “96-well dish”), there was generally no significantdifference between the different GRON chemistries in BFP to GFPconversion efficiency, especially using the 96-well plate method, asdetermined by cytometry (FIG. 1 ).

Example 3: Comparison Between 41-Mer BFP4/NC 5′-3PS/3′-3PS GRON and2′-O-Me GRONs

The purpose of this series of experiments is to compare the conversionefficiencies of the phosphothioate (PS) labeled GRONs with 3PS moietiesat each end of the GRON to 2′-O-Me or “2OMe” in the presence and absenceof a member of the bleomycin family, Zeocin™ to induce DNA breaks. Thedesigns of these GRONs are depicted in FIG. 2 . GRONs were deliveredinto Arabidopsis thaliana BFP protoplasts by PEG treatment and BFP toGFP conversion was determined at 24 h post treatment by cytometry.Samples treated with zeocin (1 mg/ml) were incubated with zeocin for 90min on ice prior to PEG treatment.

In general, the presence of zeocin (1 mg/ml) increased BFP to GFPconversion as determined by cytometry (Table 3). In both the presenceand absence of zeocin, the NC 2OMe GRON containing one 2′-O Me group onthe first RNA base at the 5′ end of the GRON was more efficacious atconverting BFP to GFP when compared to the NC 2OMe GRON containing one2′-O Me group on each of the first nine 5′ RNA bases (FIG. 2 and Table3).

In all experiments, there was no significant difference between the41-mer BFP4/NC 5′3 PS/3′3 PS and the 71-mer 2OMe BFP4/NC GRON thatcontains one 5′ 2 ′-O Me group on the first 5′ RNA base (denoted as BFP471-mer (1) NC) in BFP to GFP conversion in both the presence or absenceof 1 mg/ml of zeocin as determined by cytometry (FIG. 2 and Table 3). Itis important to note that in the presence of zeocin (and expected forbleomycin, phleomycin, tallysomycin, pepleomycin and other members ofthis family of antibiotics) that conversion becomes strand independent(i.e., both coding (C) and non-coding (NC) GRONs with the designs testedin these examples display approximately equal activity).

TABLE 3 Comparison of a standard GRON design with Okazaki fragment GRONdesigns in the presence and absence of a glycopeptide antibiotic zeocin.Exp. BFP4 41-mer BFP4 71-mer (0) BFP4 71-mer (1) BFP4 71-mer (9) Name NCC NC C NC C NC C Zeocin (+) APT043 0.13 0.0875 0.2275 0.2075 0.3550.2275 0.2325 0.195 APT066 1.9 0.713 0.762 0.683 1.318 0.7103 0.7690.883 Mean 1.015 0.40025 0.49475 0.44525 0.8365 0.4689 0.50075 0.539 StdDev 1.251579 0.442295 0.377949 0.336229 0.680944 0.341391 0.3793630.486489 SE 0.885134 0.312797 0.26729 0.237786 0.481573 0.2414360.268291 0.344052 Zeocin (−) APT043 nd nd 0.1875 0.0175 0.21 0.025 0.10.0225 APT066 0.109 0.007 0.112 0.005 0.141 0.023 0.065 0.021 Mean 0.1090.007 0.14975 0.01125 0.1755 0.024 0.0825 0.02175 Std Dev na na 0.0533870.008839 0.04879 0.001414 0.024749 0.001061 SE na na 0.037756 0.0062510.034505 0.001 0.017503 0.00075 BFP4 71-mer (0) 5′ first 10 bp are RNAand GRON has no protection NC C BFP4 71-mer (1) 5′ first 10 bp are RNAand first bp on the 5′ end has a 2′ NC C O—Me BFP4 71-mer (9) 5′ first10 bp are RNA and first nine bp on the 5′ end has a NC C 2′ O—Me

Example 4: Comparison Between 41-Mer, 101-Mer and 201-Mer BFP4/NC5′-3PS/3′-3PS GRONs

The purpose of this series of experiments was to compare the conversionefficiencies (in the presence and absence of zeocin) of thephosphothioate (PS) labeled GRONs with 3PS moieties at each end of theGRON of different lengths: 41-mer, 101-mer and 201-mer shown in Table 2.Again, the presence of zeocin (1 mg/ml) increased BFP to GFP conversionrates as determined by cytometry (Table 4). The overall trend in allthree experiments was linear with increasing NC GRON length in both thepresence and absence of zeocin. Except for the BFP-4/NC/101 andBFP-4/C/101 in the presence of zeocin, this had conversion rates thatwere close to equal but lower than the 41-mer NC GRON.

TABLE 4 Exp. BFP4 41-mer BFP4 101-mer BFP4 201-mer Name NC C NC C NC CZeocin (+) APT038 0.2425 0.1275 0.3025 0.2575 0.97 0.245 APT043 0.130.0875 0.185 0.2275 0.66 0.1875 APT047 0.3975 0.145 0.19 0.125 0.2350.085 APT052 0.3275 nd 0.17 0.21 0.585 0.225 APT052 nd nd 0.3225 0.31750.5075 0.3125 APT058 1.4275 nd 1.2 nd 1.9 nd APT066 1.9 0.713 0.992 1.051.7 0.916 Mean 0.7375 0.26825 0.480286 0.364583 0.936786 0.3285 Std Dev0.7382801 0.297475 0.428968 0.341634 0.630943 0.297412 SE 0.301461860.148738 0.162119 0.139499 0.238452 0.121442 Zeocin (−) APT038 0.05 0.010.1025 0.025 0.5725 0.025 APT066 0.109 0.007 0.214 0.047 0.566 0.035Mean 0.0795 0.0085 0.15825 0.036 0.56925 0.03 Std Dev 0.0417193 0.0021210.078842 0.015556 0.004596 0.007071 SE 0.02950446 0.0015 0.0557580.011002 0.00325 0.005001

Example 5: Delivery of Cas9 Protein into Plants

This example makes use of direct delivery of recombinant Cas9 protein toplant cells as an alternative to delivery of CRISPR-Cas expressionplasmids. This method employs carriers such as cell penetrating peptides(CPP), transfection liposome reagents, poly(ethylene glycol) (PEG),electroporation either alone or in combination to allow for delivery ofactive recombinant Cas9 protein to cells.

Methods

BFP transgenic Arabidopsis thaliana protoplasts derived from inducedroot tissue are seeded on a flat-bottom 96-well plate at 250,000 cellsper well at a cell density of 1×10⁷ cells/ml. Fluorescently-taggedrecombinant Cas9 protein (1 μg) pre-coated with CPPs at 20:1, 10: or 5:1and other CPP to cargo ratio (TAT, Penetratin, Chariot™, PEP-1 or othersfor example) or encapsulated with liposome reagents are then mixed withthe seeded protoplasts and incubated at 23° C. for 2-6 h to allow forCas9/carrier complexes to penetrate the cells. In another series oftreatments fluorescently-tagged recombinant Cas9 protein (1 μg) eitherpre-coated with CPPs as described above or not coated are introduced toprotoplasts using PEG or electorporation methodology. Protoplasts werethen analyzed by flow cytometry 24-72 h after treatment in order todetermine the percentage of Cas9 positive protoplasts within a giventreatment.

Example 6: CRISPR with 201-Mer±Wobble Base GRONs

The purpose of this series of experiments is to demonstrate BFP to GFPconversion in our Arabidopsis thaliana BFP transgenic model system usingCRISPRs to create targeted double-stranded breaks in the bfp gene andthe 201-mer GRONs to mediate conversion. The BFP CRISPR targets thecoding strand of the bfp gene and the conversion site is 27 bp upstreamof the PAM sequence (FIG. 3 ). The GRON is used as a template to repairthe double-stranded break in the bfp gene created by the CRISPR andalong with converting the targeted gene from BFP to GFP, it introduces awobble base in the bfp gene that corresponds to the PAM sequence of theBFP CRISPR as well. A wobble base in the PAM sequence of the BFP CRISPRminimizes re-cutting of the bfp gene by the CRISPRs once conversion hasoccured. This series of experiments will help to address whether or notintroducing a wobble base into the PAM sequence of the BFP CRISPR inconverted bfp genes will increase conversion efficiencies.

Methods

BFP transgenic Arabidopsis thaliana protoplasts derived from inducedroot tissue were seeded on a flat-bottom 96-well plate, at 250,000 cellsper well at a cell density of 1×107 cells/ml. The CRISPR encodedplasmids contain the mannopine synthase (MAS) promoter driving the Cas9coding sequence with an rbcSE9 terminator and the Arabidopsis U6promoter driving the sgRNA with a poly-T10 terminator. The CRISPRplasmids along with GRON were introduced into protoplasts by PEGmediated delivery at a final concentration of 0.05 μg/μl and 0.16 μMrespectively. Protoplasts were incubated in the dark at 23° C. for 72hours, and then they were analyzed by flow cytometer in order todetermine the percentage of GFP positive protoplasts within a giventreatment.

The CRISPR consists of two components: the plant codon-optimizedStreptococcus pyogenes Cas9 (SpCas9) and sgRNA both of which wereexpressed from the same plasmid. The sgRNA is a fusion of CRISPR RNA(crRNA) and trans-activating crRNA (tracrRNA). The crRNA region containsthe spacer sequence used to guide the Cas9 nuclease to the BFP targetgene (FIG. 3 ). 201-mer GRONs targeting BFP with or without wobble baseswere used to determine their effect on the rate of BFP to GFPconversion. Table 5 gives a list of the GRONs and their correspondingsequences.

TABLE 5 List of GRONs used in these examples GRON GRON Name ChemistryGRON Sequence BFP4/NC 201-mer 3 PS5′AAGATGGTGCGCTCCTGGACGTAGCCTTCGGGCATGGCGGACTTGAAGAAGTCGTGCTGCTTCATGTGGTCTGGGTAGCGGCTGAAGCACTGCACGCCGTAGGTGAAGGTGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGATGAACTTCAGGGTCAGCTTGCCGTAGGTGGCATCGCCCTCGCCC3′ (SEQ ID NO: 21) BFP4/C 201-mer 3 PS5′GGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTT3′ (SEQ ID NO: 22) BFP4/NC 201-mer 3 PS5′ AAGATGGTGCGCTCCTGGACGTAGCCTTCGGGCATGGCGGACTTGAAGAAGTCG (1 wobble)TGCTGCTTCATGTGGTCTGGGTAGCGGCTGAAGCACTGCACGCCGTAGGTGAAGGTGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGATGAACTTCAGGGTCAGCTTGCCGTAGGTGGCATCGCCCTCGCCC 3′ (SEQ ID NO: 23) BFP4/C 201-mer 3 PS5′GGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCG (1 wobble)GCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCACCTACGGCGTGCAGTGCTTCAGCCGCTACCCAGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGA GCGCACCATCTT 3′ (SEQ ID NO: 24)

Results

Using the BFP CRISPR, the BFP4/C GRON with the wobble bases is up to a3.5-fold more efficacious in BFP to GFP conversion when compared to theBFP4/C GRON without the woble bases (Table 6). There is up to a 5.9-foldincrease in BFP to GFP conversion when the BFP4/C GRON with the wobblebase is used instead of the BFP4/NC GRON with the wobble base (Table 6).Therefore, the BFP4/C GRON with the wobble base is most efficacious inBFP to GFP conversion when used with the BFP CRISPR.

Conclusions

Including a wobble base in the GRON that changes the PAM sequence of theBFP CRISPR in the converted target gene increases BFP to GFP conversion.BFP to GFP conversion by the BFP CRISPR along with the wobble-based GRONwas confirmed by Next Generation Sequencing (data not shown).Additionally, the ability of the BFP CRISPR to cleave the DNA andproduce indels in the bfp gene was confirmed by Next GenerationSequencing (data not shown).

TABLE 6 The percentage of BFP to GFP conversion as determine bycytometry at 72 h post PEG delivery of the BFP CRISPR and GRON intoprotoplasts derived from the Arabidopsis thaliana BFP transgenic line21-15-09. Percentage of GFP Positive Cells (std dev) CRISPR: BFP5 Exp.BFP4/C GRON BFP4/NC GRON Name (−) Wobbles (+) 1 wobbles (−) Wobbles (+)1 wobbles Exp 1 0.46 (0.07) 1.59 (0.06) 0.08 (0.02) 0.27 (0.04) Exp 20.24 (0.03) 0.61 (0.05) 0.04 (0.01) 0.16 (0.04)

Example 7: CRISPR with Cy3 Modified GRONs

The purpose of this series of examples is to demonstrate BFP to GFPconversion in our Arabidopsis thaliana BFP transgenic model system usingCRISPRs to create targeted double-stranded breaks in the bfp gene andGRONs to mediate conversion. The BFP6 CRISPR (CR:BFP6) used in theseexamples targets the bfp gene and causes a double-stranded break in theDNA near the site of conversion. The GRONs used with the BFP6 CRISPR,contains the coding sequence of the bfp gene around the site ofconversion and are labeled at the 5′ end with Cy3 and at the 3′ end withan idC reverse base and are herein referred to as Cy3 GRONs. These GRONsare tested at three different lengths of 41-mers, 101-mers and 201-mersand they are directly compared to the 3PS GRONs that only differ fromthe Cy3 GRONs in that they have 3 phosphothioate linkages on both the 5′and 3′ ends of the GRON. These GRONs are herein referred to as 3PSGRONs. See Table 7 for the list of GRONs used in these experiments.

Methods

BFP transgenic Arabidopsis thaliana protoplasts derived from inducedroot tissue were seeded on a flat-bottom 96-well plate, at 250,000 cellsper well at a cell density of 1×107 cells/ml. The CRISPR encodedplasmids contain the MAS promoter driving the Cas9 coding sequence withan rbcSE9 terminator and the Arabidopsis thaliana U6 promoter drivingthe sgRNA with a poly-T10 terminator. The CRISPR plasmids along withGRON were introduced into protoplasts by PEG mediated delivery at afinal concentration of 0.05 μg/μl for the CRISPR and 8.0 μM for the41-mer, 0.32 μM for the 101-mer and 0.16 μM 201-mer GRONs. GRONtreatments alone received a final GRON concentration after PEG deliveryof 8.0 μM for the 41-mer, 5.0 μM for the 101-mer and 2.5 μM for the201-mer. Protoplasts were incubated in the dark at 23° C. for 72 hours,and then they were analyzed by flow cytometer in order to determine thepercentage of GFP positive protoplasts within a given treatment.

The CRISPR consists of two components: the plant codon-optimizedStreptococcus pyogenes Cas9 (SpCas9) and sgRNA both of which wereexpressed from the same plasmid. The sgRNA is a fusion of CRISPR RNA(crRNA) and trans-activating crRNA (tracrRNA). The crRNA region containsthe spacer sequence used to guide the Cas9 nuclease to the BFP targetgene. In this experiment the BFP6 CRISPR (5′GGTGCCGCACGTCACGAAGTCGG 3′)(SEQ ID NO: 25) was used which targets the bfp gene. The GRONs containthe coding sequence of the bfp gene near the site of conversion. Table 7gives a list of the GRONs used.

TABLE 7 List of GRONs used in these examples GRON GRON Name ChemistryGRON Sequence CRISPR BFP4/C 41-mer Cy3 or 3PS5′ CCCTCGTGACCACCTTCACCTACGGCGTGCAGTGCTTCAGC BFP6 3 (SEQ ID NO: 26)′BFP4/C 101-mer Cy3 or 3PS5′CCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCACCTACGGCGTG BFP6CAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGAC 3′ (SEQ ID NO: 27)BFP4/C 201-mer Cy3 or 3PS5′GGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCA BFP6AGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTT3′ (SEQ ID NO: 28)

Results

Using the BFP6 CRISPR, the Cy3 GRONs at all lengths tested are able tomediate BFP to GFP conversion generally as efficiently as the 3PS GRONs(FIG. 4 ). Overall, the samples containing the BFP6 CRISPR and GRON havehigher levels of BFP to GFP conversion when compared to the GRON onlysamples (FIG. 4 ), demonstrating the positive impact CRISPRs have onincreasing conversion rates.

Example 8: CRISPR with GRONs of Varying Size

The purpose of this series of experiments is to demonstrate BFP to GFPconversion in an Arabidopsis thaliana BFP transgenic model system usingCRISPRs to create targeted double-stranded breaks in the bfp gene andGRONs of varying lengths to mediate conversion. The BFP CRISPR used inthese examples targets the bfp gene and causes a double-stranded breakin the DNA near the site of conversion. The GRONs used with the BFPCRISPR, contains the coding sequence of the bfp gene around the site ofconversion and are labeled at both the 5′ end and the 3′ end with 3phosphothioate linkages and are herein referred to as 3PS GRONs. TheseGRONs are tested at three different lengths of 60-mers, 101-mers and201-mers and they are directly compared to the GRON only treatments. SeeTable 8 for the list of GRONs used in these examples.

Methods

BFP transgenic Arabidopsis thaliana protoplasts derived from inducedroot tissue were seeded on a flat-bottom 96-well plate, at 250,000 cellsper well at a cell density of 1×107 cells/ml. The CRISPR encodedplasmids contain the MAS promoter driving the Cas9 coding sequence withan rbcSE9 terminator and the Arabidopsis thaliana U6 promoter drivingthe sgRNA with a poly-T10 terminator. The CRISPR plasmids along withGRON were introduced into protoplasts by PEG mediated delivery at afinal concentration of 0.05 μg/μl for the CRISPR and 0.547 μM for the60-mer, 0.32 μM for the 101-mer and 0.16 μM 201-mer GRONs. GRONtreatments alone received a final GRON concentration after PEG deliveryof 7.5 μM for the 60-mer, 5.0 μM for the 101-mer and 2.5 μM for the201-mer. Protoplasts were incubated in the dark at 23° C. for 72 hours,and then they were analyzed by flow cytometry in order to determine thepercentage of GFP positive protoplasts within a given treatment.

The CRISPR consists of two components: the plant codon-optimizedStreptococcus pyogenes Cas9 (SpCas9) and sgRNA both of which wereexpressed from the same plasmid. The sgRNA is a fusion of CRISPR RNA(crRNA) and trans-activating crRNA (tracrRNA). The crRNA region containsthe spacer sequence used to guide the Cas9 nuclease to the BFP targetgene. The BFP CRISPR spacer sequence is 5′GTCGTGCTGCTTCATGTGGT3′ (SEQ IDNO: 29). In this example the BFP CRISPR was used which targets the bfpgene. The GRONs contain the coding sequence of the bfp gene near thesite of conversion. Table 8 gives a list of the GRONs used.

TABLE 8List of GRONs used in these examples. (SEQ ID NOS: 30, 31 and 24, respectively)GRON GRON Name Chemistry GRON Sequence BFP4/C 60-mer 3 PS5′GTGACCACCTTCACCTACGGCGTGCAGTGCTTCAGCCGCTACCCAGACCACATGAAGCAG 3′(1 wobble) BFP4/C 101-mer 3 PS5′CCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCACCTACGGCGTGCAG(1 wobble) TGCTTCAGCCGCTACCCAGACCACATGAAGCAGCACGAC 3′ 3FP4/C 201-mer3 PS 5′ GGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCG(1 wobble)GCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCACCTACGGCGTGCAGTGCTTCAGCCGCTACCCAGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGA GCGCACCATCTT 3′

Results

Using the BFP CRISPR, GRONs at lengths≥101 nt are better at mediatingBFP to GFP conversion when directly compared to the 60 nt long GRONs(FIG. 5 ). Overall, the samples containing the BFP CRISPR and GRON havehigher levels of BFP to GFP conversion when compared to the GRON onlysamples (FIG. 5 ), demonstrating the positive impact CRISPRs have onincreasing conversion rates. This data further demonstrates that thelength of the GRON that is most efficacious in mediating BFP to GFPconversion, when used along with the CRISPR, needs to be ≥101 nt inlength.

Example 9: CRISPR with 2′-O-Me GRONs

The purpose of these experiments is to demonstrate BFP to GFP conversionin our Arabidopsis thaliana BFP transgenic model system using CRISPRs tocreate targeted double-stranded breaks in the bfp gene and GRONs tomediate conversion. The BFP CRISPR used in this example targets the bfpgene and causes a double-stranded break in the DNA near the site ofconversion. The GRONs used with the BFP CRISPR, contain either thecoding or non-coding sequence of the bfp gene around the site ofconversion with the first ten 5′ bases of the GRON being RNA basesinstead of DNA bases. These RNA bases are labeled with 2′-O-Me group(s)at either the first 5′ RNA base or the first nine 5′ RNA bases asdepicted in FIG. 6 . These GRONs are herein referred to as 2′-O-Me GRONsand are directly compared to the 3PS GRONs of similar lengths thatcontain DNA bases with 3 phosphothioate linkages on both the 5′ and 3′ends of the GRON. These GRONs are herein referred to as 3PS GRONs. SeeTable 9 for the list of GRONs used in these examples.

Methods

BFP transgenic Arabidopsis thaliana protoplasts derived from inducedroot tissue were seeded on a flat-bottom 96-well plate, at 250,000 cellsper well at a cell density of 1×107 cells/ml. The CRISPR encodedplasmids contain the MAS promoter driving the Cas9 coding sequence witha rbcSE9 terminator and the Arabidopsis U6 promoter driving the sgRNAwith a poly-T10 terminator. The sgRNA is a fusion of CRISPR RNA (crRNA)and trans-activating crRNA (tracrRNA). The CRISPR plasmids along withGRON were introduced into protoplasts by PEG mediated delivery at afinal concentration of 0.05 μg/μl for the CRISPR, 0.5 μM for the 71-merand 0.16 μM for the 201-mer GRONs. GRON treatments alone received afinal GRON concentration after PEG delivery of 5.0 μM for the 71-mer and2.5 μM for the 201-mer. Protoplasts were incubated in the dark at 23° C.for 72 hours, and then they were analyzed by flow cytometer in order todetermine the percentage of GFP positive protoplasts within a giventreatment.

The CRISPR consists of two components: the plant codon-optimizedStreptococcus pyogenes Cas9 (SpCas9) and sgRNA both of which wereexpressed from the same plasmid. The sgRNA is a fusion of CRISPR RNA(crRNA) and trans-activating crRNA (tracrRNA). The crRNA region containsthe spacer sequence used to guide the Cas9 nuclease to the BFP targetgene. The BFP CRISPR spacer sequence is 5′CTCGTGACCACCTTCACCCA 3′ (SEQID NO: 32). In this example the BFP CRISPR was used which targets thebfp gene. The GRONs contain either the coding or non-coding sequence ofthe bfp gene near the site of conversion. Table 9 shows a list of theGRONs used.

TABLE 9 List of GRONs used in these examples GRON GRON Name ChemistryGRON Sequence BFP4/C 71-mer 3PS5′GCUGCCCGUGCCCTGGCCCACCCTCGTGACCACCTTCACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCG3′ (SEQ ID NO: 33) BFP4/NC 71-mer 3PS5′TTCATGTGGTCGGGGTAGCGGCTGAAGCACTGCACGCCGTAGGTGAAGGTGGTCACGAGGGTGGGCCAGGG3′ (SEQ ID NO: 34) BFP4/C 201-mer 3PS5′GGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTT3′ (SEQ ID NO: 35) BFP4/NC 201-mer 3PS5′AAGATGGTGCGCTCCTGGACGTAGCCTTCGGGCATGGCGGACTTGAAGAAGTCGTGCTGCTTCATGTGGTCTGGGTAGCGGCTGAAGCACTGCACGCCGTAGGTGAAGGTGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGATGAACTTCAGGGTCAGCTTGCCGTAGGTGGCATCGCCCTCGCCC3′ (SEQ ID NO: 36) BFP4/C 71-mer 2′-O-Me5′gcugcccgugCCCTGGCCCACCCTCGTGACCACCTTCACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCG 3′ (SEQ ID NO: 37) BFP4/NC 71-mer5′uucaugugguCGGGGTAGCGGCTGAAGCACTGCACGCCGTAGGTGAAGGTGGTCACGAGGGTGGGCCAGGG3′(SEQ ID NO: 38) BFP4/C 201-mer 2′-O-Me5′gggcgagggcGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTT3′ (SEQ ID NO: 39) BFP4/NC 201-mer 2′-O-Me5′aagauggugcGCTCCTGGACGTAGCCTTCGGGCATGGCGGACTTGAAGAAGTCGTGCTGCTTCATGTGGTCGGGGTAGCGGCTGAAGCACTGCACGCCGTAGGTGAAGGTGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGATGAACTTCAGGGTCAGCTTGCCGTAGGTGGCATCGCCCTCGCCC3′ (SEQ ID NO: 40)

Results

The 71-mer and 201-mer 2′-O-Me GRONs had similar BFP to GFP conversionwhen compared to the various different types of GRON protections of (0),(1) or (9) using the BFP CRISPRs (FIGS. 7 and 8 ). The 2′-O-Me GRONs aremore efficacious than their 3PS GRON counterparts at mediating BFP toGFP conversion using the BFP CRISPRs (FIGS. 7 and 8 ).

Example 10: CRISPR Nickases with GRONs Introduction

The purpose of this example is to demonstrate BFP to GFP conversion inour Arabidopsis thaliana BFP transgenic model system using CRISPRs tocreate targeted single-stranded nicks in the bfp gene and GRONs tomediate conversion. The BFP1 CRISPR (CR:BFP1) used in this exampletargets the bfp gene and contains mutations in the catalytic residues(D10A in RuvC and H840A in HNH) that causes single-stranded nicks in theDNA of the bfp gene near the site of conversion on either the DNAstrands complementary or non-complementary to the guide RNArespectively. These CRISPRs are herein referred to as BFP1 CRISPRnickase D10A and BFP1 CRISPR nickase H840A and are used either alone orwith BFP5 sgRNA on a separate plasmid. When multiple CRISPR nickases areused together in this example, they can either nick the same DNA strandor opposite DNA strands. When both Cas9 proteins that contain the samemutations, either D10A or H840A, are used together, they nick the samestrand of DNA. Conversely, when two Cas9 proteins are used together andone of them contains the D10A mutation and the other one contains theH840A mutation, they nick opposite strands of the DNA. The GRONs usedwith the nickase CRISPRs, contains either the coding or the non-codingsequence of the bfp gene around the site of conversion with one wobblebase located in the PAM sequence of BFP5 CRISPR. These GRONs have 3phosphothioate linkages on both the 5′ and 3′ ends and are hereinreferred to as 3PS GRONs. See Table 10 for the list of GRONs used inthese experiments. The nickase CRISPRs are directly compared to theirCRISPR counterparts that are able to cause targeted double-strandedbreaks in the DNA of the bfp gene.

Methods

BFP transgenic Arabidopsis thaliana protoplasts derived from inducedroot tissue are seeded on a flat-bottom 96-well plate, at 250,000 cellsper well at a cell density of 1×107 cells/ml. The CRISPR encodedplasmids contain the MAS promoter driving the Cas9 coding sequence withan rbcSE9 terminator and the Arabidopsis thaliana U6 promoter drivingthe sgRNA with a poly-T10 terminator. The sgRNA is a fusion of CRISPRRNA (crRNA) and trans-activating crRNA (tracrRNA). The Cas9 genecontains mutations in the catalytic residues, either D10A in RuvC orH840A in HNH. The CRISPR plasmids along with GRON are introduced intoprotoplasts by PEG mediated delivery at a final concentration of 0.05μg/μl for the CRISPR and 0.16 μM for the 201-mer. GRON treatments alonereceived a final GRON concentration after PEG delivery of 2.5 μM for the201-mer. Protoplasts were incubated in the dark at 23° C. for 72 hours,and then they were analyzed by flow cytometer in order to determine thepercentage of GFP positive protoplasts within a given treatment.

The CRISPR consists of two components: the plant codon-optimizedStreptococcus pyogenes Cas9 (SpCas9) and sgRNA both of which wereexpressed from the same plasmid. The sgRNA is a fusion of CRISPR RNA(crRNA) and trans-activating crRNA (tracrRNA). The crRNA region containsthe spacer sequence used to guide the Cas9 nuclease to the BFP targetgene. In this example, the BFP1 and BFP5 sgRNA was used that targetsdifferent regions the bfp gene near the site of conversion. The BFP1spacer (5′CTCGTGACCACCTTCACCCA 3′)(SEQ ID NO: 32) targets thecoding-strand while the BFP5 spacer (5′GTCGTGCTGCTTCATGTGGT3′)(SEQ IDNO: 29) targets the non-coding strand of the bfp gene. The GRONs containeither the coding or non-coding sequence of the bfp gene near the siteof conversion. Table 10 shows a list of the GRONs used.

TABLE 10List of GRONs used in these examples (SEQ ID NOS: 41 and 42, respectively)GRON GRON Name Chemistry GRON Sequence CRISPR BFP4/NC 201-mer 3PS5′ AAGATGGTGCGCTCCTGGACGTAGCCTTCGGGCATGGCGGACTTGAAGAAGTCG BFP1 and BFP5(1 wobble; BFP5)TGCTGCTTCATGTGGTCTGGGTAGCGGCTGAAGCACTGCACGCCGTAGGTGAAGGTGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGATGAACTTCAGGGTCAGCTTGCCGTAGGTGGCATCGCCCTCGCCC 3′ BFP4/C 201-mer 3PS5′ GGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGBFP1 and BFP6 (1 wobble; BFP5)GCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCACCTACGGCGTGCAGTGCTTCAGCCGCTACCCAGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTT 3′

Results

Both of the CRISPR nickases (D10A and H840A) are more efficient atmediating BFP to GFP conversion when the BFP1 CRISPR and the BFP5 CRISPRwere used together instead of separately (FIG. 9 ). In addition, whenthe BFP1 and BFP5 D10A CRISPR nickases are used together with the C/2011W GRON, the BFP to GFP conversion is significantly higher when comparedto treatments where these CRISPR nickases are used with the NC/201 1WGRON (FIG. 9 ). When the BFP1 and BFP5 H840A CRISPR nickases are usedtogether roughly the same level of BFP to GFP conversion is observedwith either the C/201 or NC/201 1W GRONs (FIG. 9 ). These levels of BFPto GFP conversion are slightly higher than when the BFP5 CRISPR is usedalone and slightly lower than when the BFP1 CRISPR is used alone (FIG. 9).

Example 11: Use of CRISPRs to Target Multiple Genes

The purpose of this example is to demonstrate conversion of multiplegenes simultaneously in a given population of protoplasts derived fromthe Arabidopsis thaliana model system using CRISPRs to createdouble-stranded breaks in targeted genes and GRONs to mediateconversion. The CRISPRs used in this example target both the BFP andacetohydroxy acid synthase (AHAS) genes in the Arabidopsis thalianagenome by introducing into protoplasts plasmid(s) encoding the Cas9 geneand multiple sgRNA targeting these two different genes. The sgRNA is afusion of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). Thiswill allow Cas9 to cause double-stranded breaks in both the BFP and AHASgenes in the presence of GRONs that will mediate their conversion.

Methods

Arabidopsis thaliana protoplasts derived from induced root tissue areseeded on a flat-bottom 96-well plate, at 250,000 cells per well at acell density of 1×107 cells/ml. The CRISPR encoded plasmids contain theMAS promoter driving the Cas9 coding sequence with a rbcSE9 terminatorand Arabidopsis thaliana U6 promoter driving multiple different sgRNAswith a poly-T10 terminator. The sgRNA is a fusion of CRISPR RNA (crRNA)and trans-activating crRNA (tracrRNA). The CRISPR plasmids along withGRON are introduced into protoplasts by PEG mediated delivery at a finalconcentration of 0.05 μg/μl for the CRISPR and 0.16 μM for the 201-mer.GRON treatments alone receive a final GRON concentration after PEGdelivery of 2.5 μM for the 201-mer. Protoplasts will be incubated in thedark at 23° C. for 72 hours, and then they are analyzed by flowcytometer and an allele specific PCR assay in order to determine thepercentage of both BFP to GFP and AHAS converted protoplastsrespectively within a given treatment.

In the an allele specific PCR assay 10-16 replicates of 5,000 genomeequivalents of genomic DNA were used in the primary PCR reactions.

The CRISPR consists of two components: the plant codon-optimizedStreptococcus pyogenes Cas9 (SpCas9) and sgRNA both of which wereexpressed from the same or multiple plasmids. The sgRNA is a fusion ofCRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The crRNAregion contains the spacer sequence used to guide the Cas9 nuclease tothe targeted genes. In this example, different sgRNAs and GRONs are usedto target multiple genes near their sites of conversion; BFP spacer(5′CTCGTGACCACCTICACCCA 3′) (SEQ ID NO: 32) and AHAS spacer (5′TGGTIATGCAATTGGAAGATCGC 3′)(SEQ ID NO: 43). Table 11 describes the GRONsused.

TABLE 11List of GRONs used in this example (SEQ ID NOS: 44 and 45, respectively)Target GRON GRON Name Gene Chemistry GRON Sequence BFP/C 201-mer BFP 3PS5′GGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTT3′ AHAS(W) AHAS 3PS5′AGCTGCTGCAAACAGCAACATGTTCGGGAATATCTCGTCCTCCTGAGCCGGATC 574/NC 201-merCCCGAGAAATGTGTGAGCTCGGTTAGCTTTGTAGAAGCGATCTTCCAATTGCATAACCATGCCAAGATGCTGGTTGTTTAATAAAAGTACCTTCACTGGAAGATTCTCTACACGAATAGTGGCTAGCTCTTGCACATTCATTATAAA3′

Results

BFP to GFP and AHAS conversion was determined at 144 h post PEG deliveryof the BFP and AHAS CRISPR plasmids and the BFP/C 201-mer andAHAS(W)574/NC 201-mer GRONs into the Arabidopsis thaliana BFP transgenicline. Flow cytometry data revealed that Treatment 1 resulted in 0.20%BFP to GFP conversion (Table 12). Allele specific PCR assay revealedthat Treatment 1 resulted in 0.01% AHAS converted protoplasts (Table12). GRON only treatments had minimal conversion using both assays(Table 12). This example demonstrates the successful simultaneousconversion of two independent target genes (BFP and AHAS) within a givenpopulation of protoplasts derived from the Arabidopsis thaliana BFPmodel system.

TABLE 12 Measurement of conversion of both the BFP and AHAS genes at 144h post PEG delivery of CRISPR plasmids and GRONs into a given populationof protoplasts derived from the Arabidopsis thaliana BFP model systemeither by: (1) flow cytometry which determines the percentage of GFPpositive protoplasts or (2) allele specific PCR which determines thepercentage of AHAS converted protoplasts. AHAS- BFP W574L to GFPConversion conversion Allele Flow Specific Treatment CRISPR GRONsCytometry PCR 1 CR-BFP and CR-AHAS BFP/C 201-mer and AHAS(W)574/NC201-mer 0.20% ~0.01%  2 None BFP/C 201-mer and AHAS(W)574/NC 201-mer0.01% ~0.001% 3 None None 0.01% ~0.001%

Example 12: Delivery of Cas9 mRNA into Plant Cells

This example makes use of direct delivery of recombinant Cas9 mRNA intoplant cells as an alternative to delivery of CRISPR-Cas expressionplasmids. This method includes (1) in vitro synthesis of modified mRNAand (2) Delivery of this modified mRNA into plant cells.

Methods

A Cas9 mRNA will be transcribed in vitro using an RNA polymerase such asT7, T3 or SP6 from a linearized plasmid template including components ofa 5′UTR, the coding sequence (CDS) for the protein and a 3′UTR. One RNApolymerase may incorporate a particular modified nucleoside better thananother. The 5′ UTR may contain elements that improve its stability suchas the MiR-122 of hepatitis C virus (Shimakami et al., 2012). In vitrosynthesis will incorporate nucleosides that are protective and ensuregood translation in the target plant cells. Recombinant Cas9 mRNA willbe capped and contain a polyA tail.

BFP transgenic Arabidopsis thaliana protoplasts derived from inducedroot tissue are seeded on a flat-bottom 96-well plate at 250,000 cellsper well at a cell density of 1×107 cells/ml. Recombinant Cas9 mRNA willbe delivered into plant cells in one of the following means (list notinclusive): cell penetrating peptides (CPP), transfection liposomereagents, poly(ethylene glycol) (PEG) or electroporation either alone orin combination to allow for delivery of active recombinant Cas9 mRNAinto cells.

Example 13: CRISPR-Cas for Tethering DNA, RNA or Proteins

This example makes use of a modified single guide RNA (sgRNA) cassettewherein a linker sequence (may also be referred to as a tetheringsequence) is included at the 3′ end of the tracrRNA but 5′ of the RNApolymerase III termination signal as shown in the example below (FIG. 10). The sgRNA is a fusion of CRISPR RNA (crRNA) and trans-activatingcrRNA (tracrRNA). Though preferred, the placement of the linker is notlimited to the 3′ end of the tracerRNA but will be investigated atseveral positions within the sgRNA cassette. The linker sequence mayvary in nucleotide length or contain secondary structure that wouldimprove tethering or increase the number of molecules tethered throughtriplex interactions.

The linker will allow for Watson-Crick base pairing with a DNA, RNA orproteins that contains the complementary sequence (FIG. 10 ).Additionally, linker sequence in sgRNA cassettes will be designed tocontain advanced secondary and tertiary structure allowing for morecomplex multifaceted interaction regions that would tether multiple DNA,RNA or protein molecules.

The overall concept is that a CRISPR-Cas complex will tether biologicalmolecules to the site of nuclease activity thereby increasing thelikelihood of gene editing. These biological molecules include the GRONwhich will mediate conversion of targeted gene(s). Tethering linkers canbe added to sgRNA by simply using, for example, Gene Strings or annealedoligos.

Methods

BFP transgenic Arabidopsis thaliana protoplasts derived from inducedroot tissue are seeded on a flat-bottom 96-well plate, at 250,000 cellsper well at a cell density of 1×107 cells/ml. The CRISPR-Cas tetheringplasmids contains the MAS promoter driving the Cas9 coding sequence witha rbcSE9 terminator and

Arabidopsis thaliana U6 promoter driving sgRNAs with a linker sequencethat is complementary to a polynucleotide tract of 15-30 bp located on a201-mer editing GRON targeting bfp (as shown in FIG. 10 ). The sgRNAtethering cassette is terminated by a poly-T10 terminator. TheCRISPR-Cas plasmids along with GRON are introduced into protoplasts byPEG mediated delivery at a final concentration of 0.05 μg/μl for theCRISPR and 0.16 μM of the 201-mer GRON. GRON treatments alone received afinal GRON concentration after PEG delivery of 2.5 μM for the 201-mer.Protoplasts were incubated in the dark at 23° C. for 72 hours, and thenthey were analyzed by flow cytometer in order to determine thepercentage of GFP positive protoplasts within a given treatment.

The CRISPR consists of two components: the plant codon-optimizedStreptococcus pyogenes Cas9 (SpCas9) and sgRNA both of which areexpressed from the same or different plasmids. The sgRNA is a fusion ofCRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) and linker. ThecrRNA region contains the spacer sequence used to guide the Cas9nuclease to the BFP target gene. In this example CRISPR is used whichtargets the bfp gene. The GRONs contain either the coding or non-codingsequence of the bfp gene near the site of conversion.

Example 14: CRISPRs with Truncated gRNA

The purpose of this example is to demonstrate conversion of BFP to GFPin protoplasts derived from the Arabidopsis thaliana BFP model systemusing CRISPRs to create double-stranded breaks in targeted genes andGRONs to mediate conversion. The CRISPRs used in this example targetsthe bfp gene in the Arabidopsis thaliana genome by introducing intoprotoplasts plasmid(s) encoding the Cas9 gene and one sgRNAs that is twodifferent lengths. The sgRNA is a fusion of CRISPR RNA (crRNA) andtrans-activating crRNA (tracrRNA). The crRNA which guides the Cas9 tothe target genes is called the spacer and it is typically 20-nt inlength (CR:BFP1 20-nt), however, in these examples we tested theeffectiveness of using a smaller length spacer of 17-nt (CR:BFP1 17-nt)in mediating BFP to GFP conversion.

Methods

Arabidopsis thaliana protoplasts derived from induced root tissue areseeded on a flat-bottom 96-well plate, at 250,000 cells per well at acell density of 1×107 cells/ml. The CRISPR encoded plasmids contain theMAS promoter driving the Cas9 coding sequence with a rbcSE9 terminatorand Arabidopsis thaliana U6 promoter driving multiple different sgRNAswith a poly-T10 terminator. The sgRNA is a fusion of CRISPR RNA (crRNA)and trans-activating crRNA (tracrRNA). The CRISPR plasmids along withGRON are introduced into protoplasts by PEG mediated delivery at a finalconcentration of 0.05 μg/μl for the CRISPR and 0.16 μM for the 201-mer.GRON treatments alone receive a final GRON concentration after PEGdelivery of 2.5 μM for the 201-mer. Protoplasts will be incubated in thedark at 23° C. for 72 hours, and then they are analyzed by flowcytometer in order to determine the percentage of BFP to GFP within agiven treatment.

The CRISPR consists of two components: the plant codon-optimizedStreptococcus pyogenes Cas9 (SpCas9) and sgRNA both of which wereexpressed from the same or multiple plasmids. The sgRNA is a fusion ofCRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The crRNAregion contains the spacer sequence used to guide the Cas9 nuclease tothe targeted genes. In these examples, two different length BFP1 spacersof 20-nt (5′CTCGTGACCACCTTCACCCA 3′) (SEQ ID NO: 32) vs. 17-nt(5′GTGACCACCTTCACCCA 3′)(SEQ ID NO: 46) were tested. Table 13 describesthe GRON used

TABLE 13 List of GRON used in this example (SEQ ID NO: 47) GRON NameGRON Chemistry GRON Sequence BFP4/NC 3PS5′AAGATGGTGCGCTCCTGGACGTAGCCTTCGGGCATGGCGGACTTGAAGAAGTCG 201-mer 3WTGCTGCTTCATGTGGTCGGGGTAGCGGCTGAAGCACTGCACGCCGTACGTAAACGTGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGATGAACTTCAGGGTCAGCTTGCCGTAGGTGGCATCGCCCTCGCCC 3′

Results

Reducing the length of the BFP1 protospacer from 20 bp to 17 bp hadsimilar levels of BFP to GFP conversion of 0.163% vs. 0.177%respectively at 72 h post PEG delivery of plasmids and GRONs into theArabidopsis thaliana BFP model system (FIG. 11 ).

Example 15: CRISPRs with Amplicon gRNA

The purpose of this example is to demonstrate conversion of BFP to GFPin protoplasts derived from the Arabidopsis thaliana BFP model systemusing CRISPRs to create double-stranded breaks in targeted genes andGRONs to mediate conversion. The CRISPRs used in this example targetsthe bfp gene in the Arabidopsis thaliana genome by introducing intoprotoplasts plasmid(s) encoding the Cas9 gene and one sgRNAs that iseither encoded on a plasmid or introduced into protoplasts as anamplicon. The sgRNA is a fusion of CRISPR RNA (crRNA) andtrans-activating crRNA (tracrRNA). The crRNA guides the Cas9 to thetarget genes, where Cas9 creates a double-stranded break and the GRON isused as a template to convert BFP to GFP in a site-directed manner.

Methods

Arabidopsis thaliana protoplasts derived from induced root tissue areseeded on a flat-bottom 96-well plate, at 250,000 cells per well at acell density of 1×10⁷ cells/mil. The CRISPR encoded plasmids contain theMAS promoter driving the Cas9 coding sequence with a rbcSE9 terminatorand Arabidopsis U6 promoter driving multiple different sgRNAs with apoly-T₁₀ terminator. The sgRNA is a fusion of CRISPR RNA (crRNA) andtrans-activating crRNA (tracrRNA). The CRISPR plasmids along with GRONare introduced into protoplasts by PEG mediated delivery at a finalconcentration of 0.05 μg/μl for the CRISPR and 0.16 μM for the 201-mer.GRON treatments alone receive a final GRON concentration after PEGdelivery of 2.5 μM for the 201-mer. Protoplasts will be incubated in thedark at 23° C. for 72 hours, and then they are analyzed by flowcytometer in order to determine the percentage of BFP to GFP within agiven treatment.

The CRISPR consists of two components: the plant codon-optimizedStreptococcus pyogenes Cas9 (SpCas9) and sgRNA both of which wereexpressed from the same or multiple plasmids. The sgRNA is a fusion ofCRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The crRNAregion contains the spacer sequence used to guide the Cas9 nuclease tothe targeted genes. In these examples, the same BFP6 gRNA(5′GGTGCCGCACGTCACGAAGTCGG 3′) (SEQ ID NO: 25) was delivered intoprotoplasts either as an amplicon or encoded on a plasmid. Table 14describes the GRONs used.

TABLE 14List of GRONs used in this example (SEQ ID NOS: 48 and 49, respectively)GRON GRON Name Chemistry GRON Sequence BFP4/NC 201-mer 3PS5′AAGATGGTGCGCTCCTGGACGTAGCCTTCGGGCATGGCGGACTTGAAGAAGTCGTGCTGCTTCATGTGGTCTGGGTAGCGGCTGAAGCACTGCACGCCGTAGGTGAAGGTGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGATGAACTTCAGGGTCAGCTTGCCGTAGGTGGCATCGCCCTCGCCC3′ BFP4/C 201-mer 3PS5′GGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTT3′

Results

Delivery of the BFP6 gRNA as an amplicon (CR:BFP6 (gRNA amplicon)) alongwith a plasmid containing only Cas9 had similar rates of BFP to GFPconversion when compared to treatments with both the gRNA (gRNA plasmid)and Cas9 being encoded on separate plasmids at 72 h post PEG delivery ofplasmids and GRONs into the Arabidopsis thaliana BFP model system (FIG.12 ).

Example 16: CRISPRs with Unmodified GRONs

The purpose of this example is to demonstrate BFP to GFP conversion inour Arabidopsis thaliana BFP transgenic model system using CRISPRs tocreate targeted double-stranded breaks in the bfp gene and GRONs tomediate conversion. The BFP CRISPR used in this example targets the bfpgene and causes a double-stranded break in the DNA near the site ofconversion. The 3PS GRONs contain DNA bases with 3 phosphothioatelinkages on both the 5′ and 3′ ends of the GRON and are herein referredto as 3PS GRONs. The 3PS GRONs were directly compared to theirunmodified GRON counterparts in mediated BFP to GFP conversion using theBFP CRISPRs in our BFP transgenic Arabidopsis thaliana model system. SeeTable 15 for the list of GRONs used in these examples

TABLE 15List of GRONs used in this example (SEQ ID NOS: 50 and 51, respectively)GRON Name GRON Chemistry GRON Sequence BFP4/C 41-mer 3PS5′CCCTCGTGACCACCTTCACCTACGGCGTGCAGTGCTTCAGC 3′ BFP4/NC 41-mer None5′GCTGAAGCACTGCACGCCGTAGGTGAAGGTGGTCACGAGGG3′

Methods

BFP transgenic Arabidopsis thaliana protoplasts derived from inducedroot tissue were seeded on a flat-bottom 96-well plate, at 250,000 cellsper well at a cell density of 1×107 cells/ml. The CRISPR encodedplasmids contain the MAS promoter driving the Cas9 coding sequence witha rbcSE9 terminator and the Arabidopsis U6 promoter driving the sgRNAwith a poly-T10 terminator. The sgRNA is a fusion of CRISPR RNA (crRNA)and trans-activating crRNA (tracrRNA). The CRISPR plasmids along withGRON were introduced into protoplasts by PEG mediated delivery at afinal concentration of 0.05 μg/μl for the CRISPR, 0.16 μM for the 41-merGRONs. GRON treatments alone received a final GRON concentration afterPEG delivery of 0.8 μM for the 41-mer. Protoplasts were incubated in thedark at 23° C. for 72 hours, and then they were analyzed by flowcytometer in order to determine the percentage of GFP positiveprotoplasts within a given treatment.

The CRISPR consists of two components: the plant codon-optimizedStreptococcus pyogenes Cas9 (SpCas9) and sgRNA both of which wereexpressed from the same plasmid. The sgRNA is a fusion of CRISPR RNA(crRNA) and trans-activating crRNA (tracrRNA). The crRNA region containsthe spacer sequence used to guide the Cas9 nuclease to the BFP targetgene. The BFP CRISPR spacer sequence is 5′CTCGTGACCACCTTCACCCA 3′(SEQ IDNO: 32). In this example the BFP CRISPR was used which targets the bfpgene. The GRONs contain the non-coding sequence of the bfp gene near thesite of conversion. Table 16 shows a list of the GRONs used

TABLE 16List of GRONs used in this example. (SEQ ID NOS: 50 and 51, respectively)GRON Name GRON Chemistry GRON Sequence BFP4/C 41-mer 3PS5′CCCTCGTGACCACCTTCACCTACGGCGTGCAGTGCTTCAGC 3′ BFP4/NC 41-mer None5′GCTGAAGCACTGCACGCCGTAGGTGAAGGTGGTCACGAGGG3′

Results

The 41-mer 3PS GRONs are more efficacious than their unmodified GRONcounterparts at mediating BFP to GFP conversion using the BFP CRISPRs(FIG. 13 ).

Example 17: TALENs and GRONs in Flax

The purpose of this example is to demonstrate EPSPS conversion in flaxat both 24 hours in protoplasts and 3-weeks in microcalli after deliveryof TALEN plasmids and GRONs. The TALENs used in this example targets theepsps gene in the Linum usitatissimum genome by introducing into shoottip derived protoplasts plasmid(s) encoding TALENs creates adouble-stranded break and the GRON is used as a template to convert theepsps gene in a site-directed manner.

Methods

Flax protoplasts were isolated from shoot tips obtained from in vitrogerminated seedlings. The TALEN plasmids along with GRONs wereintroduced into protoplasts by PEG mediated delivery at a finalconcentration of 0.05 μg/μl and 0.5 μM respectively. Protoplasts wereincubated in the dark at 25° C. for up to 48 h in liquid medium, orembedded in alginate beads (5×10⁵ cells/ml), and cultured in liquidmedium to induce cell division and the formation of microcalli.Protoplasts or microcalli samples obtained 24 h or 3 weeks after DNAdelivery were analyzed by NGS to determine the percentage of cells (DNAreads) carrying out the target mutation within a given treatment. Thepercent of indels generated by imperfect NHEJ-mediated DNA repair wasalso estimated.

TALEN constructs include two arms (left and right), each consisting of aTAL effector-like DNA binding domain linked to a catalytic DNA cleavagedomain of FokI. The TAL effector-like DNA binding domain guides theTALEN arms to specific sites of DNA which allows the FokI endonucleasesof each arm to dimerize together and cleave double-stranded DNA. TheTALEN encoded plasmids contains MasP::LuEPSPS_(Leftarm)-T2A-LuEPSPS_(right arm) with a rbcSE9 terminator. LuEPSPS_(leftarm) sequence is 5′TGGAACAGCTATGCGTCCG 3′ (SEQ ID NO: 52) and theLuEPSPS_(right arm) sequence is 5′TGAGTTGCCTCCAGCGGCT 3′ (SEQ ID NO:53). GRONs (144-mers) targeting LuEPSPS with or without wobble baseswere used to determine their effect on rate of conversion.

Results

24 hour protoplasts and 3-week old microcalli have 0.067% and 0.051%EPSPS conversion respectively as determined by Next GenerationSequencing (FIG. 14 ). Additionally, these data show that the TALEN isactive and able to cleave the epsps target gene in Linum usitatissimumand form indels of 2.60% and 1.84% respectively at 24 hours inprotoplasts and up to 3-week in microcalli. Moreover, EPSPS conversionand indels are maintained up to 3 weeks after the TALEN plasmid and GRONare introduced.

Example 18: CRISPRs and GRONs in Flax

The purpose of this example is to demonstrate activity of Cas9 in flaxmicrocalli three and six weeks after delivery of a Cas9 plasmid. TheCRISPRs used in this example targets the epsps gene in the Linumusitatissimum genome by introducing into shoot tip derived protoplastsplasmid(s) encoding the Cas9 gene and a sgRNAs. The sgRNA is a fusion ofCRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The crRNAguides the Cas9 to the target genes, where Cas9 creates adouble-stranded break in the epsps gene in a site-directed manner. Thedouble-stranded breaks in the epsps gene when repaired by the ubiquitousNHEJ pathway will cause indels to form around the cleavage site.

Methods

Flax protoplasts were isolated from shoot tips obtained from in vitrogerminated seedlings. The CRISPR encoded plasmids contains the MASpromoter driving the Cas9 coding sequence with an rbcSE9 terminator andthe Arabidopsis thaliana U6 promoter driving the sgRNA with a poly-T₁₀terminator. The CRISPR plasmids were introduced into protoplasts by PEGmediated delivery at a final concentration of 0.05 μg/μl. Protoplastswere embedded in alginate beads (5×10⁵ cells/ml), cultured in liquidmedium, and incubated in a rotatory shaker (30 rpm) in the dark at 25°C. Microcalli developed from individual cells were analyzed by NGS, 3and 6 weeks after CRISPR plasmid delivery, to determine the percentageof cells (DNA reads) carrying out indels generated by the error-proneNHEJ-mediated DNA repair pathway.

The CRISPR consists of two components: the plant codon-optimizedStreptococcus pyogenes Cas9 (SpCas9) and sgRNA both of which wereexpressed from the same plasmid. The sgRNA is a fusion of CRISPR RNA(crRNA) and trans-activating crRNA (tracrRNA). The crRNA region containsthe spacer sequence used to guide the Cas9 nuclease to the target gene.In this example, the CRISPR targets the epsps gene.

Results

3- and 6-week old microcalli have 46.5% and 54.7% indel formationrespectively as determined by Next Generation Sequencing (FIG. 15 ).These data shows that Cas9 is active and able to cleave the EPSPS targetgene in Linum usitatissimum and form indels. Moreover, these indels aremaintained up to 6 weeks after the CRISPR plasmid was introduced.

Example 19: Construction of Engineered Nucleases

CRISPR-Cas

For construction of transient CRISPR-Cas9 expression plasmids, a higherplant codon-optimized SpCas9 gene containing a SV40 NLS at both the N-and C-terminal and a 2×FLAG tag on the N-terminal was synthesized as aseries of GeneArt® Strings™ (Life Technology, Carlsbad, Calif.), thencloned downstream of the mannopine synthase (MAS) promoter and upstreamof the pea ribulose bisphosphate carboxylase (rbcsE9) terminator byGibson's method. Next, an sgRNA cassette consisting of a chimeric gRNAwhose expression is driven by the Arabidopsis U6 promoter, wassynthesized as GeneArt® Strings™, then shuttled into the Cas9 containingconstruct using Gibson's method forming pBCRISPR. To specify thechimeric gRNA for the respective target sequence, pairs of DNAoligonucleotides encoding the variable 20-nt sgRNA targeting sequenceswere annealed to generate short double strand fragments with 4-bpoverhangs. The fragments were ligated into BbsI digested pBCrispr toyield CRISPR-Cas constructs BC-1, BC-2 and BC-3.

TALEN

Design and construction of TALEN expression constructs BT-1 and LuET-1was based on rules as described in Cermak et al., Nucleic Acids Res. 39,e82 (2011). The target sequence was selected based on the gene editingsite and the repeat variable i-residue (RVD) following the rules thatNG, HD, NI, and NN recognize T, C, A, and G, respectively. The assemblyof TAL effector domain linked to the heterodimeric FokI domains wascompleted through a commercial service (GeneArt; Life Technologies).TALEN monomers were cloned downstream of the MAS promoter and upstreamof the rbcE9 terminator using Gibson's method and expressed as a 2Acoupled unit.

Cell Culture and Protoplast Isolation

Surface-sterilized Arabidopsis seeds were germinated on solid ½ MSmedium (MS medium containing half the concentration of minerals andvitamins; 87.7 mM sucrose; Murashige and Skoog, 1962) at 25° C. under a12 h light/dark cycle. Root material from 2 to 3-week-old seedlings werecollected and maintained in MS liquid medium under low light conditionsat 28° C. Root cultures were transferred and maintained in MSAR[0.22% ½MS, 87.7 mM sucrose, 11.4 μM IAA, 2.3 μM 2,4-D, 1.5 μM 2iP, pH 5.8]three weeks prior to protoplast isolation. Roots were cut intoapproximately 6 mm segments and incubated in MSAP solution [0.22% ½ MS,87.7 mM sucrose, 11.4 μM IAA, 2.3 μM 2,4-D, 1.5 μM 2iP, and 400 mMmannitol, pH 5.8] containing cell wall digesting enzymes [1.25%Cellulase RS, 0.25% Macerozyme R-10, 0.6 M mannitol, 5 mM MES, 0.1% BSA]for 3-4 h in the dark with gentle shaking. The released protoplasts werecollected and passed through a sterile 100 μm filter and 35 μm filter.The protoplast filtrate was mixed with 0.8 times the volume of Optiprep™Density Gradient Medium (Sigma) and mixed gently. A 60% W5 [154 mM NaCl,5 mM KCl, 125 mM CaCl₂.2H₂O, 5 mM glucose, 10 mM MES, (pH 5.8)]/40%Optiprep solution followed by 90% W5/10% Optiprep solution were slowlylayered onto the filtrate/Optiprep solution to make a gradient, whichwas centrifuged at 198 RCF for 10 min. The white protoplast layer wascollected and mixed with 2 times the volume of W5. Protoplasts werecentrifuged at 44 RCF for 10 min and re-suspended in TM solution [14.8mM MgCl₂.6H₂O, 5 mM MES, 572 mM mannitol, (pH 5.8)] at a density of 1×10cells/ml. For experiments with Zeocin™ (Life Technologies, Carlsbad,Calif.) and phleomycin (InvivoGen, San Diego, Calif.), protoplasts werekept in TM adjusted to pH 7.0 for 90 min on ice before transfection.

Flax protoplasts were isolated from shoot tips obtained from 3-week-oldseedlings germinated in vitro. Shoot tips were finely chopped with ascalpel, pre-plasmolyzed for 1 h at room temperature in B-medium [B5salts and vitamins (Gamborg et al., 1968), 4 mM CaCl₂, 0.1 M glucose,0.3 M mannitol, 0.1 M glycine, 250 mg/l casein hydrolysate, 10 mg/lL-cystein-HCL, 0.5% polyvinylpyrrolidone (MW 10,000), 0.1% BSA, 1 mg/lBAP, 0.2 mg/l NAA, and 0.5 mg/l 2,4-D], and incubated in a cell walldigesting enzyme solution containing B-medium supplemented with 0.66%Cellulase YC and 0.16% Macerozyme R-10 over a rotatory shaker (50 rpm)at 25° C. for 5 h. Released protoplasts were sieved and purified bydensity gradient centrifugation using Optiprep (Sigma) layers, countedwith a hemocytometer, and kept stationary overnight in the dark at adensity of 0.5×10⁶ protoplasts/ml in B medium.

Protoplast Transfection

In a 96-well flat bottom plate, 2.5×10⁵ cells per well were transfectedwith 10 pmol GRON, 10 pmol GRON plus 3.25 μg CRISPR-Cas or TALENexpression construct or mock using PEG [270 mM mannitol, 67.5 mMCa(NO₃)₂, 38.4% PEG 1500, (pH 5.8)]. Cells and DNA were incubated withPEG on ice for 30 minutes followed by a wash with 200 μl of W5 solution.Finally, 85 μl of MSAP++[MSAP containing 50 nM phytosulfokine-α and 20μM n-propyl gallate] was added and the cells cultured in low lightconditions at 28° C.

After about 18 h of culture, protoplasts were transfected with TALENplasmid along with GRONs (20 μg plasmid and 0.2 nmol GRON/10⁶protoplasts) using PEG mediated delivery. Treated protoplasts wereincubated in the dark at 25° C. for up to 48 h in B medium, or embeddedin alginate beads 24 h after transfection, and cultured in V-KM liquidmedium to induce cell division and the formation of microcalli. For theantibiotic experiments, 1.25×10⁵ cells per well were transfected with 8μM GRON CG13 using the PEG solution described above.

Cytometry

Seventy-two h after transfection, cells were analyzed by cytometry usingthe Attune® Acoustic Focusing cytometer (Applied Biosystems®) withexcitation and detection of emission as appropriate for GFP. Backgroundlevel was based on PEG-treated protoplasts without DNA delivery. Forantibiotic experiments, protoplasts treated with Zeocin or phleomycinprior to transfection were analyzed by cytometry 48 h aftertransfection.

Sequencing Analysis

Genomic DNA was extracted from CRISPR-Cas or TALEN-treated protoplastsusing the NucleoSpin® Plant II kit as per the manufacturer'srecommendations (Machery-Nagel, Bethlehem, Pa.). Amplicons flanking theTALEN or CRISPR target region were generated using Phusion® polymerasewith 100 ng of genomic DNA and primers BFPF-1(5′-GGACGACGGCAACTACAAGACC-3′)(SEQ ID NO: 54)/BFPR-1(5′-TAAACGGCCACAAGTrCAGC-3′) (SEQ ID NO: 55) for Arabidopsis CRISPR andTALEN; or LuEPF-1 (5′-GCATAGCAGTGAGCAGAAGC-3′) (SEQ ID NO: 56)/LuEPR-15′-AGAAGCTGAAAGGCTGGAAG-3′ (SEQ ID NO: 57) for L. usitatissimum TALEN.The amplicons were purified and concentrated using Qiaquick MinElutecolumns (Qiagen, Valencia, Calif.). Deep sequencing of the amplicons wasperformed by GeneWiz (South Plainfield, N.J.) using a 2×250 bp MiSeq run(Illumina, San Diego, Calif.). For data analysis fastq files for readland read 2 were imported into CLC Genomics Workbench 7.0.4 (CLCBio,Boston, Mass.). Paired reads were merged into a single sequence if theirsequences overlapped. A sequence for an amplicon was identified if it orits reverse and complemented sequence contained both forward and reverseprimer sequences. Occurrence of a unique sequence in a sample wasrecorded as its abundance. Percent indel or gene edit was calculated bydividing the number of reads with the edit or indel by the total numberof sequenced reads, and then multiplying by 100.

Sequence of CRISPR-Cas photospacers (SEQ ID NOS: 58-60, respectively)Name Sequence (5′ to 3′) Figure Reference BC-1 CTCGTGACCACCTTCACCCA1a; 1b; 2a; 2c BC-2 GTCGTGCTGCTTCATGTGG 2b BC-3 GGCTGAAGCACTGCACGCCG 2dTALEN binding domain sequences (SEQ ID NOS: 61-64, respectively)Paper Name Sequence (5′ to 3′) Figure Reference BT-1Left arm: TGGTCGGGGTAGCGGCTGA 3a; 3b Right arm: TCGTGACCACCTTCACCCALuET-1 Left arm: TGGAACAGCTATGCGTCCG 3c; 3dRight arm: TGAGTTGCCTCCAGCGGCTGRON sequences used (SEQ ID NOS: 65-78, respectively) NameSequence (5′ to 3′) Chemistry Figure Reference CG1GCTGAAGCACTGCACGCCGTAGGTGAAGGTGGTCACGAGGG Unmodified 2a CG2G*C*T*GAAGCACTGCACGCCGTAGGTGAAGGTGGTCACGA*G*G*G (*) = 3PS 2a CG3C*G*C*TCGTGACCACCTTCACCTACGGCGTGCAGTGCTTC*A*G*C (*) = 3PS 2d; 3a CG4VCCCTCGTGACCACCTTCACCTACGGCGTGCAGTGCTTCAGCH V = CY3; H = 3′DMT dC 2d CG5G*T*G*ACCACCTTCACCTACGGCGTGCAGTGCTTCAGCCGCTACCCAGACCACATGAAG*C*A*G(*) = 3PS 2b CG6A*A*G*ATGGTGCGCTCCTGGACGTAGCCTTCGGGCATGGCGGACTTGAAGAAGTCGTGCTGCTTCATGTGGTCGGGGTAG(*) = 3PS 1b; 2cCGGCTGAAGCACTGCACGCCGTAGGTGAAGGTGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGATGAACTTCAGGGTCAGCTTGCCGTAGGTGGCATCGCCCTCG*C*C*C CG7G*G*G*CGAGGGCGATGCCACCTACGGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGC(*) = 3PS 3bCCACCCTCGTGACCACCTTCACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGA GCGCACCAT*C*T*T CG8G*G*G*CGAGGGCGATGCCACCTACGGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGC(*) = 3PS 2bCCACCCTCGTGACCACCTTCACCTACGGCGTGCAGTGCTTCAGCCGCTACCCAGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGA GCGCACCAT*C*T*T CG9A*A*G*ATGGTGCGCTCCTGGACGTAGCCTTCGGGCATGGCGGACTTGAAGAAGTCGTGCTGCTTCATGTGGTCGGGGTAG(*) = 3PS 1aCGGCTGAAGCACTGCACGCCGTACGTAAACGTGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGATGAACTTCAGGGTCAGCTTGCCG TAGGTGGCATCGCCCTCG*C*C*C CG10(a)agauggugcGCTCCTGGACGTAGCCTTCGGGCATGGCGGACTTGAAGAAGTCGTGCTGCTTCATGTGGTCGGGGTAGCGLower Case = RNA 2cGCTGAAGCACTGCACGCCGTAGGTGAAGGTGGTCACGAGGGTGGGCCAGGGCACGGGGCAGCTTGCCGGTGGTGCAGATbases; (base) = 2′-O-Me; GAACTTCAGGGTCAGCTTGCCGTAGGTGGCATCGCCCTCGCCCUpper Case = DNA bases CG11(a)(a)(g)(a)(u)(g)(g)(u)(g)cGCTCCTGGACGTAGCCTTCGGGCATGGCGGACTTGAAGAAGTCGTGCTGCTTCATGTGGTCGGLower Case = RNA 2cGGTAGCGGCTGAAGCACTGCACGCCGTAGGTGAAGGTGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGbases; (base) = 2′-O-Me;TGCAGATGAACTTCAGGGTCAGCTTGCCGTAGGTGGCATCGCCCTCGCCCUpper Case = DNA bases CG12VCGGTCGGTAAACTGGCGAAGAACGATATTGAACTTTTCCTTGGAAATGCTGGAATAGCTATGCGTGCGCTGACAGCTGCTV = CY3; H = ′DMT dC 3c; 3dGTAACAGCCGCTGGAGGGCAACTCAAGGTCCCTTCCCTCAACTCCTTCCAGCCTTTCAGCTTCTTH andCPGVCGGTCGGTAAACTGGCGAAGAACGATATTGAACTTTTCCTTGGAAATGCTGGAATAGCTATGCGTGCGCTGACAGCTGCTGTAACAGCCGCTGGAGGGCAACTCAAGGTCCCTTCCCTCAACTCCTTCCAGCCTTTCAGCTTCTTH CG13G*C*T*GAAGCACTGCACGCCGTGGGTGAAGGTGGTCACGA*G*G*G (*) = 3PSExtended Data FIG. 1

Statistical Analysis

Statistical significance was determined using a Student's t-test withtwo-tailed distribution. P-values<0.05 were considered as significant.Data are shown as mean and SEM.

Results

CRISPR-Cas Nuclease Activity and Gene Editing in A. thalianaProtoplasts.

Engineered nucleases such as TAL effector nucleases (TALENs) andclustered regularly interspaced short palindromic repeats(CRISPR)-associated endonuclease Cas9 (CRISPR-Cas9) can be programmed totarget and cleave double-stranded DNA with high specificity. TALENsconsist of two arms, both having a TAL effector-like DNA binding domain(TALE) linked to a catalytic DNA nuclease domain of FokI. The TALEdomains guide the TALEN arms to specific sites of DNA allowing fordimerization of the FokI endonucleases and subsequent generation of adouble strand break (DSB). The CRISPR-Cas9 system consists of a twocomponents; a Streptococcus pyogenes Cas9 nuclease (SpCas9) and achimeric fusion of two RNAs (crRNA and tracrRNA) referred to as anengineered single guide (sgRNA). The sgRNA supports targeted nucleicacid specificity for Cas9 through base pairing of its first twenty 5′bases with the DNA target, subsequently resulting in a site-specificDSB.

In plants, DSBs are typically repaired by the error prone non-homologousend joining (NHEJ) DNA repair pathway resulting in random deletionsand/or insertions (indels) at the site of repair. In contrast precisiongenome editing, relies on nuclease induced DSBs near the targeted changeto be repaired by homology directed repair (HDR), a repair pathway thatis more precise than NHEJ due to the requirement of a homologoustemplate DNA-in most cases sister chromatid. By harnessing the HDRpathway, it is possible to use an engineered oligonucleotide as therepair template to edit DNA specifically, when cleaved by nucleases ornon-specifically when used in combination with double strand breakinducing antibiotics.

FIG. 16 depicts CRISPR-Cas9 nuclease activity in Arabidopsis thalianaprotoplasts derived from the BFP transgenic model system in which astably integrated BFP gene can be converted to GFP by editing the codonencoding H66 (CAC→TAC H66Y). When cells were treated with CRISPR-Cas9(BC-1), NHEJ induced indels were produced at a frequency of 0.79% nearthe H66 locus of the BFP gene by deep sequencing (FIG. 16 a ). Themajority of indels were single bp and none longer than 9 bp. Conversely,cells treated with GRON only or mock-treated did not exhibit indels(data not shown). These results show that CRISPR-Cas9 nuclease canactively target the BFP gene in this transgenic model system.

With regard to the effectiveness of CRISPR-Cas9 in combination with GRONto mediate BFP to GFP gene editing in protoplasts derived from ourtransgenic model system, a 7.4-fold improvement in BFP to GFP editingwas observed when both CRISPR-Cas9 and phosphorothioate (PS) modifiedGRONs (CG6) are introduced concurrently when compared to GRON alone orCRISPR-Cas9 alone treatments (FIG. 16 b ). These results demonstratethat introducing CRISPR-Cas9 with PS modified GRONs into Arabidopsisprotoplasts significantly increase the frequency of BFP to GFP geneediting.

GRONs containing three adjacent PS modifications (herein refer to as3PS) at both the 5′ and 3′ ends positively effect BFP to GFP editingwhen compared to an unmodified GRON. The 3PS modified GRON (CG2), whencombined with CRISPR-Cas9 (BC-1), is more efficacious at BFP to GFPediting when compared to an unmodified GRON template (CG1; FIG. 17 a ).In addition, a positive correlation between editing and GRON length(FIG. 17 b ) was observed. Taken together, these results show that bothGRON modification and length can greatly improve the frequency of geneediting in plants such as Arabidopsis in the presence of CRISPR-Cas9.

When either the 201 nucleobase (nb) 3PS modified GRON (CG6), or the 201nb 2′-O-methyl modified GRONs (CG9) or (CG10), consisting of the firstten 5′ bases as RNA with either the first RNA base or the first 9 RNAbases modified with 2′-O-methyl are introduced along with CRISPR-Cas9(BC-1) into Arabidopsis protoplasts, no statistical difference in BFP toGFP editing between them was observed (FIG. 17 c ). Similarly, wheneither the 201 nb 3PS modified GRON (CG3) or the 201 nb Cy3 modifiedGRON (CG4), comprising of a 5′ Cy3 and an 3′ idC reverse base, wereintroduced along with CRISPR-Cas9 (BC-3) into Arabidopsis protoplasts,no statistical difference in editing frequencies was observed (FIG. 17 d). Overall, these data show that diverse GRON modifications can greatlyimprove the frequency of gene editing in Arabidopsis in the presence ofCRISPR-Cas9.

Based on these results with CRISPR-Cas9 and modified GRONs, it wasdetermined if modified GRONs coupled with TALEN pairs targeting the BFPgene result in improved BFP to GFP gene editing as well. To first showeffective nuclease activity at the BFP locus Arabidopsis protoplastswere treated with TALENs (BT-1) and found 0.51% indels at or near theexpected cleavage site by deep sequencing-indicating that TALENs are asactive as CRISPR-Cas9 in this model system (FIG. 18 a ). The majority ofdeletions were >10 bp but less than 50 bp, while insertions,significantly less abundant than deletions were three bp or less. Nextwe examined the effectiveness of TALENs coupled with modified GRON toedit BFP to GFP. A 9.2-fold improvement in the frequency of BFP to GFPediting was observed when both the BFP TALEN (BT-1) and 3PS GRON (CG7)are introduced when compared to 3PS GRON alone (FIG. 18 b ). Similar tothe CRISPR-Cas experiments described above, these results demonstratethat introducing TALENs with 3PS modified GRONs into Arabidopsisprotoplasts also significantly increases the frequency of BFP to GFPgene editing.

The EPSPS (5′-enolpyruvylshikimate-3-phosphate synthase) loci in Linumusitatissimum (Common flax) was also used as a target in this system.The EPSPS genes encode an enzyme in the shikimate pathway thatparticipates in the biosynthesis of the aromatic amino acidsphenylalanine, tyrosine and tryptophan. In plants, EPSPS is a target forthe herbicide, glyphosate, where it acts as a competitive inhibitor ofthe binding site for phosphoenolpyruvate.

TALENs targeting a site near two loci (T97I and P101A) in L.usitatissimum that when edited will render EPSPS tolerant to glyphosatewere selected. Delivering TALEN (LuET-1) together with a 144 nb 5′ Cy3modified GRON (CG11) containing the targeted changes at T97I and P101Ainto protoplasts, gene editing frequencies of 0.19% at both loci andindel frequency at 0.5% seven days after introduction were observed(FIG. 18 c, 18 d ). The majority of indels were 10 bp or less (FIG. 18 c). These results demonstrate that introducing TALENs with Cy3 modifiedGRONs into L. usitatissimum protoplasts significantly increase thefrequency of EPSPS gene editing and further that multiple nucleotideedits can be realized with a single GRON.

Example 20: Effect of Two Members of the Bleomycin Family of Antibioticson Conversion

The purpose of this series of examples was to evaluate the effect ofantibiotics on conversion efficiencies.

Methods

Protoplasts from an Arabidopsis thaliana line with multiple copies of ablue fluorescent protein gene were treated with GRON as described inExample 1, with the following modification: before the addition of GRON,the protoplasts were kept for 90 minutes on ice in a solution of TM(14.8 mM MgCl₂×6H₂O, 5 mM 2-(N-morpholino)ethanesulfonic acid, 572 mMmannitol), supplemented with 0, 250, or 1000 μg/ml Zeocin™ orphleomycin. The pH of the solutions was adjusted to 7.0. The percentageof green-fluorescing cells resulting from BFP to GFP conversion wasevaluated by flow cytometry as described in Example 1.

Results

Zeocin and phleomycin at both concentrations used (250 and 1000 μg/ml)resulted in an increase in BFP to GFP gene editing (see FIG. 19 ).Green-fluorescing cells resulting from BFP to GFP gene editing wereobserved five days after GRON delivery (FIG. 20 ).

REFERENCES

-   1. LeCong et al 2013 Science: vol. 339 no. 6121 pp. 819-823.-   2. Jinek et al 2012 Science. 337:816-21-   3. Wang et al 2008 RNA 14: 903-913-   4. Zhang et al 2013. Plant Physiol. 161: 20-27

Example 21: CRISPRs and GRONs in Rice

The purpose of this experiment is to demonstrate ACCase conversion inOryza sativa at 120 hours after PEG delivery of CRISPR-Cas plasmids andGRONs into protoplasts. The CRISPR-Cas used in this experiment targetsthe accase gene in the rice genome by introducing into protoplastsplasmid(s) encoding the Cas9 gene and a sgRNAs. The sgRNA is a fusion ofCRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The crRNAguides the Cas9 to the target genes, where Cas9 creates adouble-stranded break in the accase gene and the GRON is used as atemplate to convert the accase gene in a site-directed manner.

Methods

Rice protoplasts were isolated from calli. The CRISPR-Cas encodedplasmids contains the corn ubiquitin promoter driving the Cas9 codingsequence with an rbcSE9 terminator and a rice U6 promoter promoterdriving the sgRNA with a poly-T₁₀ terminator. The CRISPR-Cas plasmidswere introduced into protoplasts by PEG mediated delivery at a finalconcentration of 0.05 μg/μl. GRONs with the following sequence, 5′ V CTGA CCT GAA CTT GAT CTC AAT TAA CCC TG CGG TTC CAG AAC ATT GCC TTr TGCAGT CCT CTC AGC ATA GCA CTC AAT GCG GTC TGG GTT TAT CTT GCT TCC AAC GACAAC CCA AGC CCC TCC TCG TAG CTC TGC AGC CAT GGG AAT GTA GAC AAA GGC AGGCTG ATT GTA TGT CCT AAG GTT CTC AAC AAT AGT CGA GCC H 3′ (SEQ ID NO:79), were used at a final concentration of 0.8 μM. Protoplasts wereembedded in agarose (2.5×106 cells/ml), cultured in liquid medium, andincubated in a rotatory shaker (60 rpm) in the dark at 28° C. Individualsamples were analyzed by NGS, 120 hours after CRISPR-Cas plasmid and/orGRON delivery, to determine the percentage of cells (DNA reads) carryingthe ACCase conversion and having indels in the accase gene.

The CRISPR consists of two components: the plant codon-optimizedStreptococcus pyogenes Cas9 (SpCas9) and sgRNA both of which wereexpressed from the same plasmid. The sgRNA is a fusion of CRISPR RNA(crRNA) and trans-activating crRNA (tracrRNA). The crRNA region containsthe spacer sequence (5′-ACGAGGAGGGGCTTGGGTTGTGG-3)(SEQ ID NO: 80) usedto guide the Cas9 nuclease to the target gene. In this experiment theCRISPR targets the accase gene.

Results

At 120 h, rice protoplasts have 0.026% ACCase conversion as determinedby Next Generation Sequencing. GRON only controls with no CRISPR-Casshowed minimal Accase conversion of 0.002% at 120 hours and theuntreated controls showed no conversion. Additionally, these data showthat the CRISPR-Cas is active and able to cleave the ACCase target geneand form indels of 8.0%.

Example 22: CRISPRs and GRONs in Rice

Summary:

Targeted ACCase mutations have been identified in nineteen week-oldcalli by PCR and DNA sequencing analyses.

Introduction

The purpose of this experiment is to demonstrate ACCase conversion inOryza sativa calli after PEG delivery of CRISPR-Cas plasmids and GRONsinto protoplasts. The CRISPR-Cas used in this experiment targets theACCase gene in the rice genome by introducing into protoplastsplasmid(s) encoding the Cas9 gene and a sgRNAs. The sgRNA is a fusion ofCRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The crRNAguides the Cas9 to the target gene, where Cas9 creates a either adouble-stranded break or a nick at a targeted location in the ACCasegene and the GRONs are used as a template to convert the ACCase gene ina site-directed manner.

Results

Targeted OsACCase mutations described in the tables below, have beenidentified in nineteen week-old calli by PCR and DNA sequencinganalyses.

Methods

Rice protoplasts were isolated from suspension cultures initiated frommature seed-derived embryogenic calli. The CRISPR-Cas plasmids withGRONs at a final concentration of 0.05 μg/μl and 0.8 μM, respectively,were introduced into protoplasts by PEG mediated delivery method. Anexemplary range for CRISPR-Cas plasmids at a final concentrationinclude, but is not limited to 0.01 to 0.2 μg/μl. An exemplary range forGRON at a final concentration include, but is not limited to 0.01 to 4μM. The CRISPR-Cas encoded plasmids contains the corn ubiquitin promoterdriving the Cas9 coding sequence with an rbcSE9 terminator and a rice U6promoter promoter driving the sgRNA with a poly-T₁₀ terminator. Sequenceinformation of the GRONs are described in Table 3. Following the PEGtreatment, protoplasts were embedded in agarose (1.25×10⁶ cells/ml),cultured in liquid medium, and incubated in a rotatory shaker (60 rpm)in the dark at 28° C. An exemplary range for embedding protoplasts inagarose include, but is not limited to 0.625×10⁶ to 2.5×10⁶ cells/ml.Samples from each treatment were analyzed by Next Generation Sequencingafter 4 weeks post CRISPR-Cas plasmid and/or GRON treatment to determinethe proportion of cells (DNA reads) carrying the ACCase conversion.Microcalli from converted treatments were released onto solid selectionmedium containing clethodim (0.25-0.5 μM) or sethoxydim (2.5 μM).Individual callus lines growing on this selection medium after 19 weeksof culture were analyzed by in-house screening methods as well as DNAsequencing in order to identify individual calli containing the targetedACCase conversions.

The CRISPR consists of two components: the Cas9, such as a plantcodon-optimized Streptococcus pyogenes Cas9 (SpCas9) and sgRNA both ofwhich were expressed from plasmid(s). Cas9 and sgRNA can also bedelivered by mRNA/RNA or protein/RNA respectively. The sgRNA is a fusionof CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The crRNAregion contains the spacer with sequences described in Table 2, whichwere used to guide the Cas9 nuclease to the target gene. In thisexperiment the CRISPR targets the rice ACCase gene.

List of conversions in OsACCase at two different locations within thegene, Site 1 and Site 2. For each site, all combinations of conversionevents are possible.

OsACCase Site1 Site 2 I1781A D2078G I1781L D2078K I1781M D2078T I1781NS2079F I1781S K2080E I1781T C2088F I1781V C2088G G1783C C2088H A1786PC2088K C2088L C2088N C2088P C2088Q C2088R C2088S C2088T C2088V C2088W

List of CRISPR-Cas gRNA spacer sequences used in this experiment. Spacerlength may vary up to ±20 bp. Mismatched within the spacer sequence canbe tolerated up to 10 bp.

OsACCase SEQ ID Sample ID Spacer RNA Sequence (5′ to 3′) Site 1 Site 281 1 CAUAAGAUGCAGCUAGACAG X 82 2 AGCUAGACAGUGGUGAAAUU X 83 3UAGACAGUGGUGAAAUUAGG X 84 4 AGACAGUGGUGAAAUUAGGU X 85 5GUGGGUUAUUGAUUCUGUUG X 86 6 UGGGUUAUUGAUUCUGUUGU X 87 7UAUUGAUUCUGUUGUGGGCA X 88 8 UCUGUUGUGGGCAAGGAAGA X 89 9GUGGGCAAGGAAGAUGGACU X 90 10 CAAGGAAGAUGGACUUGGUG X 91 11CUUGGUGUGGAGAAUAUACA X 92 12 CUAUUGCCAGUGCUUAUUCU X 93 13UGCUUAUUCUAGGGCAUAUA X 94 14 UUUACACUUACAUUUGUGAC X 95 15UUUGUGACUGGAAGAACUGU X 96 16 ACUGGAAGAACUGUUGGAAU X 97 17GGAGCUUAUCUUGCUCGACU X 98 18 AUAUGCCCUAGAAUAAGCAC X 99 19GAUAAGAUGCAGCUAGACAG X 100 20 GGCUAGACAGUGGUGAAAUU X 101 21GAGACAGUGGUGAAAUUAGG X 102 22 GGACAGUGGUGAAAUUAGGU X 103 24GGGGUUAUUGAUUCUGUUGU X 104 25 GAUUGAUUCUGUUGUGGGCA X 105 26GCUGUUGUGGGCAAGGAAGA X 106 28 GAAGGAAGAUGGACUUGGUG X 107 29GUUGGUGUGGAGAAUAUACA X 108 30 GUAUUGCCAGUGCUUAUUCU X 109 31GGCUUAUUCUAGGGCAUAUA X 110 32 GUUACACUUACAUUUGUGAC X 111 33GUUGUGACUGGAAGAACUGU X 112 34 GCUGGAAGAACUGUUGGAAU X 113 36GUAUGCCCUAGAAUAAGCAC X 114 37 CGACUAUUGUUGAGAACCUU X 115 38UGCCUUUGUCUACAUUCCCA X 116 39 CCCAUGGCUGCAGAGCUACG X 117 40AUGGCUGCAGAGCUACGAGG X 118 41 UGGCUGCAGAGCUACGAGGA X 119 42GGCUGCAGAGCUACGAGGAG X 120 43 CAGAGCUACGAGGAGGGGCU X 121 44AGAGCUACGAGGAGGGGCUU X 122 45 ACGAGGAGGGGCUUGGGUUG X 123 46GCAUUGAGUGCUAUGCUGAG X 124 47 UAUGCUGAGAGGACUGCAAA X 125 48GACUGCAAAAGGCAAUGUUC X 126 49 GGCAAUGUUCUGGAACCGCA X 127 50GCAAUGUUCUGGAACCGCAA X 128 51 GGUUAAUUGAGAUCAAGUUC X 129 52UGAGAUCAAGUUCAGGUCAG X 130 53 GUUCAGGUCAGAGGAACUCC X 131 54AACUCCAGGAUUGCAUGAGU X 132 55 CAAGCCGACUCAUGCAAUCC X 133 56UUGAUCUCAAUUAACCCUUG X 134 57 UCUCAGCAUAGCACUCAAUG X 135 58GCAUAGCACUCAAUGCGGUC X 136 59 CAUAGCACUCAAUGCGGUCU X 137 60UCCUCGUAGCUCUGCAGCCA X 138 61 AGCCAUGGGAAUGUAGACAA X 139 62AUGGGAAUGUAGACAAAGGC X 140 63 CAGGCUGAUUGUAUGUCCUA X 141 64GGACUAUUGUUGAGAACCUU X 142 65 GGCCUUUGUCUACAUUCCCA X 143 66GCCAUGGCUGCAGAGCUACG X 144 67 GUGGCUGCAGAGCUACGAGG X 145 68GGGCUGCAGAGCUACGAGGA X 146 69 GAGAGCUACGAGGAGGGGCU X 147 70GGAGCUACGAGGAGGGGCUU X 148 71 GCGAGGAGGGGCUUGGGUUG X 149 72GAUGCUGAGAGGACUGCAAA X 150 73 GGAGAUCAAGUUCAGGUCAG X 151 74GACUCCAGGAUUGCAUGAGU X 152 75 GAAGCCGACUCAUGCAAUCC X 153 76GUGAUCUCAAUUAACCCUUG X 154 77 GCUCAGCAUAGCACUCAAUG X 155 78GAUAGCACUCAAUGCGGUCU X 156 79 GCCUCGUAGCUCUGCAGCCA X 157 80GGCCAUGGGAAUGUAGACAA X 158 81 GUGGGAAUGUAGACAAAGGC X 159 82GAGGCUGAUUGUAUGUCCUA X

A list of GRON sequences suitable for use in this experiment areprovided in the table below (V=CY3; H=3′DMT dC CPG).

SEQ ID 160 1 VGTCATAGCACATAAGATGCAGCTAGACAGTGGTGAAATTAGGTGGGTTATTGATTCTGTTGTGGGCAAGGAAGATGGACTTGGTGTGGAGAATGCTCATGGAAGTGCTGCTATTGCCAGTGCTTATTCTAGGGCATATAAGGAGACATTTACACTTACATTTGTGACTGGAAGAACTGTTGGAATAGGAGCTTATCTTGCTCH 161 2VGAGCAAGATAAGCTCCTATTCCAACAGTTCTTCCAGTCACAAATGTAAGTGTAAATGTCTCCTTATATGCCCTAGAATAAGCACTGGCAATAGCAGCACTTCCATGCAGATTCTCCACACCAAGTCCATCTTCCTTGCCCACAACAGAATCAATAACCCACCTAATTTCACCACTGTCTAGCTGCATCTTATGTGCTATGACH 162 3VGTCATAGCACATAAGATGCAGCTAGACAGTGGTGAAATTAGGTGGGTTATTGATTCTGTTGTGGGCAAGGAAGATGGACTTGGTGTGGAGAATATACATTGCAGTGCTGCTATTGCCAGTGCTTATTCTAGGGCATATAAGGAGACATTTACACTTACATTTGTGACTGGAAGAACTGTTGGAATAGGAGCTTATCTTGCTCH 163 4VGAGCAAGATAAGCTCCTATTCCAACAGTTCTTCCAGTCACAAATGTAAGTGTAAATGTCTCCTTATATGCCCTAGAATAAGCACTGGCAATAGCAGCACTGCAATGTATATTCTCCACACCAAGTCCATCTTCCTTGCCCACAACAGAATCAATAACCCACCTAATTTCACCACTGTCTAGCTGCATCTTATGTGCTATGACH 164 5VGTCATAGCACATAAGATGCAGCTAGACAGTGGTGAAATTAGGTGGGTTATTGATTCTGTTGTGGGCAAGGAAGATGGACTTGGTGTGGAGAATATACATGGAAGTGCTCCAATTGCCAGTGCTTATTCTAGGGCATATAAGGAGACATTTACACTTACATTTGTGACTGGAAGAACTGTTGGAATAGGAGCTTATCTTGCTCH 165 6VGAGCAAGATAAGCTCCTATTCCAACAGTTCTTCCAGTCACAAATGTAAGTGTAAATGTCTCCTTATATGCCCTAGAATAAGCACTGGCAATGGTAGCACTTCCATGTATATTCTCCACACCAAGTCCATCTTCCTTGCCCACAACAGAATCAATAACCCACCTAATTTCACCACTGTCTAGCTGCATCTTATGTGCTATGACH 166 7VCTGACCTGAACTTGATCTCAATTAACCCTTGCGGTTCCAGAACATTGCCTTTTGCAGTCCTCTCAGCATAGCACTCAATGCGGTCTGGGTTTATCTTGCTTCCAACGACAACCCAAGCCCCTCCTCGTAGCTCTGCAGCCATGGGAATGTAGACAAAGGCAGGCTGATTGTATGTCCTAAGGTTCTCAACAATAGTCGAGCCH 167 8VGGCTCGACTATTGTTGAGAACCTTAGGACATACAATCAGCCTGCCTTTGTCTACATTCCCATGGCTGCAGAGCTACGAGGAGGGGCTTGGGTTGTGGTTGGTAGCAAGATAAACCCAGACCGCATTGAGTGCTATGCTGAGAGGACTGCAAAAGGCAATGTTCTGGAACCGCAAGGGTTAATTGAGATCAAGTTCAGGTCAGH 168 9VCTGACCTGAACTTGATCTCAATTAACCCTTGCGGTTCCAGAACATTGCCTTTTGCAGTCCTCTCAGCATAGCACTCAATGCGGTCTGGGTTTATCTTAAAATCAACGACAACCCAAGCCCCTCCTCGTAGCTCTGCAGCCATGGGAATGTAGACAAAGGCAGGCTGATTGTATGTCCTAAGGTTCTCAACAATAGTCGAGCCH 169 10VGGCTCGACTATTGTTGAGAACCTTAGGACATACAATCAGCCTGCCTTTGTCTACATTCCCATGGCTGCAGAGCTACGAGGAGGCGCTTGGGTTGTGGTTGATAGCAAGATAAACCCAGACCGCATTGAGAGGTATGCTGAGAGGACTGCAAAAGGCAATGTTCTGGAACCGCAAGGGTTAATTGAGATCAAGTTCAGGTCAGH 170 11VGGCTCGACTATTGTTGAGAACCTTAGGACATACAATCAGCCTGCCTTTGTCTACATTCCCATGGCTGCAGAGCTACGAGGAGGGGCTTGGGTTGTGGTTGATAGCGAAATAAACCCAGACCGCATTGAGTGCTATGCTGAGAGGACTGCAAAAGGCAATGTTCTGGAACCGCAAGGGTTAATTGAGATCAAGTTCAGGTCAGH 171 12VCTGACCTGAACTTGATCTCAATTAACCCTTGCGGTTCCAGAACATTGCCTTTTGCAGTCCTCTCAGCATAGCACTCAATGCGGTCTGGGTTTATTTCGCTATCAACCACAACCCAAGCGCCTCCTCGTAGCTCTGCAGCCATGGGAATGTAGACAAAGGCAGGCTGATTGTATGTCCTAAGGTTCTCAACAATAGTCGAGCCH 172 13VGGCTCGACTATTGTTGAGAACCTTAGGACATACAATCAGCCTGCCTTTGTCTACATTCCCATGGCTGCAGAGCTACGAGGAGGGGCTTGGGTTGTGGTTGATAGCAAGATAAACCCAGACCGCATTGAGCGTTATGCTGAGAGGACTGCAAAAGGCAATGTTCTGGAACCGCAAGGGTTAATTGAGATCAAGTTCAGGTCAGH 173 14VCTGACCTGAACTTGATCTCAATTAACCCTTGCGGTTCCAGAACATTGCCTTTTGCAGTCCTCTCAGCATATTGCTCAATGCGGTCTGGGTTTATCTTGCTATCAACGACAACCCAAGCCCCTCCTCGTAGCTCTGCAGCCATGGGAATGTAGACAAAGGCAGGCTGATTGTATGTCCTAAGGTTCTCAACAATAGTCGAGCCH

Example 23: CRISPRs and GRONs in Flax

Summary:

Targeted LuEPSPS mutations have been identified in four week-old calliby PCR and DNA sequencing analyses.

Introduction

The purpose of this experiment is to demonstrate conversion of the EPSPSgenes in the Linum usitatissimum genome in shoot tip derived protoplastsby PEG mediated delivery of CRISPR-Cas plasmids and GRONs. TheCRISPR-Cas and GRONs used in this experiment target the EPSPS genes inthe flax genome. The CRISPR consists of two components: a Cas9, such asa plant codon-optimized Streptococcus pyogenes Cas9 (SpCas9) and sgRNAboth of which are expressed from plasmid(s). Cas9 and sgRNA can also bedelivered by mRNA/RNA or protein/RNA respectively. The sgRNA is a fusionof CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The crRNAguides the Cas9 to the targeted genes, where Cas9 creates a either adouble-stranded break or nick in the EPSPS genes and the GRONs are usedas a template to convert the EPSPS genes in a site-directed manner.

Results

Targeted LuEPSPS (T97I and/or the P101A, P101T or P101S and/or the G96A)mutations have been identified in four week-old calli by PCR and DNAsequencing analyses. Shoots have been regenerated from these convertedcalli.

Methods

Flax protoplasts were isolated from shoot tips obtained from in vitrogerminated seedlings. The CRISPR-Cas encoded plasmids contain the MASpromoter driving the Cas9 coding sequence with an rbcSE9 terminator andthe Arabidopsis thaliana U6 promoter driving the sgRNA with a poly-T₁₀terminator. The CRISPR-Cas plasmids were introduced into protoplasts byPEG mediated delivery at a final concentration of 0.05 μg/μl. GRONstargeting each of the two flax LuEPSPS genes (Table 2) were used at afinal concentration of 4.0 μM. An exemplary range for CRISPR-Casplasmids at a final concentration include, but is not limited to 0.01 to0.2 μg/μl. An exemplary range for GRON at a final concentration include,but is not limited to 0.01 to 4 μM. Protoplasts were embedded inalginate beads (5×10⁵ cells/ml), cultured in liquid medium, andincubated in a rotatory shaker (30 rpm) in the dark at 25° C. Anexemplary range for embedding protoplasts in alginate beads include, butis not limited to 3.0×10⁵ to 7.5×10⁵ cells/ml. Microcalli developed fromindividual cells were analyzed by Next Generation Sequencing, 3 and 7weeks after CRISPR-Cas plasmid and GRON delivery, to determine theproportion of cells (DNA reads) carrying the targeted mutations in theLuEPSPS genes. Larger calli were grown from 8-week-old convertedmicrocalli plated over solid regeneration medium, and shoots starteddifferentiating from regenerated calli after about 4-8 weeks. Convertedcalli and shoots with the targeted EPSPS gene mutations were identifiedby PCR and DNA sequencing analyses.

The CRISPR consists of two components: the plant codon-optimizedStreptococcus pyogenes Cas9 (SpCas9) and sgRNA both of which wereexpressed from plasmid(s). The sgRNA is a fusion of CRISPR RNA (crRNA)and trans-activating crRNA (tracrRNA). The crRNA region contains thespacer with sequences described in the table below, which were used toguide the Cas9 nuclease to the EPSPS targeted genes.

List of CRISPR-Cas gRNA spacer sequences used in this experiment. Spacerlength may vary up to ±20 bp. Mismatched within the spacer sequence canbe tolerated up to 10 bp.

LuEPSPS Spacer RNA Genes SEQ ID Sample ID Sequence (5′ to 3′) 1 2 174 1CAGAAGCGCGCCAUUGUUGA X X 175 2 CGCGCCAUUGUUGAAGGUUG X 176 3CGCGCCAUUGUUGAAGGUCG X 177 4 GCCAUUGUUGAAGGUUGUGG X 178 5GCCAUUGUUGAAGGUCGUGG X 179 6 AGGUUGUGGUGGUGUGUUUC X 180 7AGGUCGUGGUGGUGUGUUUC X 181 8 UGUGGUGGUGUGUUUCCGGU X 182 9CGUGGUGGUGUGUUUCCGGU X 183 10 UGUGUUUCCGGUCGGUAAAC X X 184 11UGUUUCCGGUCGGUAAACUG X 185 12 AACGAUAUUGAACUUUUCCU X 186 13AACGAUAUCGAACUUUUCCU X 187 14 GAACUUUUCCUUGGAAAUGC X X 188 15ACAGCUGCUGUAACAGCCGC X X 189 16 GCUGCUGUAACAGCCGCUGG X X 190 17AACUCAAGCUACAUACUCGA X 191 18 AACUCAAGCUACAUACUCGA X 192 19CGAAUGAGAGAGAGACCAAU X 193 20 CGAAUGAGAGAGAGACCGAU X 194 21AGAGAGACCAAUUGGAGAUU X 195 22 CCAAUUGGAGAUUUGGUUGU X 196 23CCGAUUGGAGAUUUAGUUGU X 197 24 CCAACAACCAAAUCUCCAAU X 198 25CCAACAACUAAAUCUCCAAU X 199 26 AUUGGUCUCUCUCUCAUUCG X 200 27AUCGGUCUCUCUCUCAUUCG X 201 28 GUAGCUUGAGUUGCCUCCAG X X 202 29GCUGUUACAGCAGCUGUCAG X X 203 30 UAGCUGUUCCAGCAUUUCCA X X 204 31UUCUUCGCCAGUUUACCGAC X 205 32 UUCUUCCCCAGUUUACCGAC X 206 33ACCACCACAACCUUCAACAA X 207 34 ACCACCACGACCUUCAACAA X 208 35GAGAAGCGCGCCAUUGUUGA X X 209 36 GGCGCCAUUGUUGAAGGUUG X 210 37GGCGCCAUUGUUGAAGGUCG X 211 38 GGGUUGUGGUGGUGUGUUUC X 212 39GGGUCGUGGUGGUGUGUUUC X 213 40 GGUGGUGGUGUGUUUCCGGU X 214 41GGUGGUGGUGUGUUUCCGGU X 215 42 GGUGUUUCCGGUCGGUAAAC X X 216 43GGUUUCCGGUCGGUAAACUG X 217 44 GACGAUAUUGAACUUUUCCU X 218 45GACGAUAUCGAACUUUUCCU X 219 46 GCAGCUGCUGUAACAGCCGC X X 220 47GACUCAAGCUACAUACUCGA X 221 48 GACUCAAGCUACAUACUCGA X 222 49GGAAUGAGAGAGAGACCAAU X 223 50 GGAAUGAGAGAGAGACCGAU X 224 51GGAGAGACCAAUUGGAGAUU X 225 52 GCAAUUGGAGAUUUGGUUGU X 226 53GCGAUUGGAGAUUUAGUUGU X 227 54 GCAACAACCAAAUCUCCAAU X 228 55GCAACAACUAAAUCUCCAAU X 229 56 GUUGGUCUCUCUCUCAUUCG X 230 57GUCGGUCUCUCUCUCAUUCG X 231 58 GAGCUGUUCCAGCAUUUCCA X X 232 59GUCUUCGCCAGUUUACCGAC X 233 60 GUCUUCCCCAGUUUACCGAC X 234 61GCCACCACAACCUUCAACAA X 235 62 GCCACCACGACCUUCAACAA X

A list of GRON sequences suitable for use in this experiment areprovided in the table below (V=CY3; H=3′DMT dC CPG).

SEQ ID 236 1 VCGGTCGGTAAACTGGCGAAGAACGATATTGAACTTTTCCTTGGAAATGCTGCTATAGCTATGCGTGCGCTGACAGCTGCTGTAACAGCCGCTGGAGGCAACTCAAGGTCCCTTCCCTCAACTCCTTCCAGC CTTTCAGCTTCTTH 237 2VAAGAAGCTGAAAGGCTGGAAGGAGTTGAGGGAAGGGACCTTGAGTTGCCTCCAGCGGCTGTTACAGCAGCTGTCAGCGGACGCATAGCTGTGGCAGCATTTCCAAGGAAAAGTTCAATATCGTTCTTCGC CAGTTTACCGACCGH 238 3VCGGTCGGTAAACTGGCGAAGAACGATATTGAACTTTTCCTTGGAAATGCTGGAATAGCTATGCGTGCGCTGACAGCTGCTGTAACAGCCGCTGGAGGCAACTCAAGGTCCCTTCCCTCAACTCCTTCCAGCCTTTCAGCTTCTTH 239 4VAAGAAGCTGAAAGGCTGGAAGGAGTTGAGGGAAGGGACCTTGAGTTGCCTCCAGCGGCTGTTACAGCAGCTGTCAGCGCACGCATAGCTATTCCAGCATTTCCAAGGAAAAGTTCAATATCGTTCTTCGC CAGTTTACCGACCGH 240 5VCGGTCGGTAAACTGGCGAAGAACGATATTGAACTTTTCCTTGGAAATGCTGGAATTGCTATGCGTTCTCTGACAGCTGCTGTAACAGCCGCTGGAGGCAACTCAAGGTCCCTTCCCTCAACTCCTTCCAGC CTTTCAGCTTCTTH 241 6VAAGAAGCTGAAAGGCTGGAAGGAGTTGAGGGAAGGGACCTTGAGTTGCCTCCAGCGGCTGTTACAGCAGCTGTCAGTGAACGCATAGCAATTCCAGCATTTCCAAGGAAAAGTTCAATATCGTTCTTCGC CAGTTTACCGACCGH 242 7VCGGTCGGTAAACTGGCGAAGAACGATATTGAACTTTTCCTTGGAAATGCTGGAATCGCTATGCGTACTCTGACAGCTGCTGTAACAGCCGCTGGAGGCAACTCAAGGTCCCTTCCCTCAACTCCTTCCAGC CTTTCAGCTTCTTH 243 8VAAGAAGCTGAAAGGCTGGAAGGAGTTGAGGGAAGGGACCTTGAGTTGCCTCCAGCGGCTGTTACAGCAGCTGTCAGTGTACGCATAGCAATTCCAGCATTTCCAAGGAAAAGTTCAATATCGTTCTTCGC CAGTTTACCGACCGH 244 9VCGGTCGGTAAACTGGCGAAGAACGATATTGAACTTTTCCTTGGAAATGCTGGAATTGCTATGCGTGCGCTGACAGCTGCTGTAACAGCCGCTGGAGGCAACTCAAGGTCCCTTCCCTCAACTCCTTCCAGC CTTTCAGCTTCTTH 245 10VAAGAAGCTGAAAGGCTGGAAGGAGTTGAGGGAAGGAACCTTGAGTTGCCTCCAGCGGCTGTTACAGCAGCTGTCAGCGCACGCATAGCTATTCCAGCATTTCCAAGGAAAAGTTCGATATCGTTCTTCCC CAGTTTACCGACCGH 246 11VCGGTCGGTAAACTGGCGAAGAACGATATTGAACTTTTCCTTGGAAATGCTGGAATAGCTATGCGTGCTCTGACAGCTGCTGTAACAGCCGCTGGAGGCAACTCAAGGTCCCTTCCCTCAACTCCTTCCAGC CTTTCAGCTTCTTH 247 12VAAGAAGCTGAAAGGCTGGAAGGAGTTGAGGGAAGGGACCTTGAGTTGCCTCCAGCGGCTGTTACAGCAGCTGTCAGCGCACGCATAGCTGTTCCAGCATTTCCAAGGAAAAGTTCAATATCGTTCTTCGC CAGTTTACCGACCGH 248 13VCGGTCGGTAAACTGGGGAAGAACGATATCGAACTTTTCCTTGGAAATGCTGCTATAGCTATGCGTGCGCTGACAGCTGCTGTAACAGCCGCTGGAGGCAACTCAAGGTTCCTTCCCTCAACTCCTTCCAGC CTTTCAGCTTCTTH 249 14VAAGAAGCTGAAAGGCTGGAAGGAGTTGAGGGAAGGAACCTTGAGTTGCCTCCAGCGGCTGTTACAGCAGCTGTCAGCGGACGCATAGCTGTTGCAGCATTTCCAAGGAAAAGTTCGATATCGTTCTTCCC CAGTTTACCGACCGH 250 15VCGGTCGGTAAACTGGGGAAGAACGATATCGAACTTTTCCTTGGAAATGCTGGAATAGCTATGCGTGCGCTGACAGCTGCTGTAACAGCCGCTGGAGGCAACTCAAGGTTCCTTCCCTCAACTCCTTCCAGC CTTTCAGCTTCTTH 251 16VAAGAAGCTGAAAGGCTGGAAGGAGTTGAGGGAAGGAACCTTGAGTTGCCTCCAGCGGCTGTTACAGCAGCTGTCAGCGCACGCATAGCTATTCCAGCATTTCCAAGGAAAAGTTCGATATCGTTCTTCCC CAGTTTACCGACCGH 252 17VCGGTCGGTAAACTGGGGAAGAACGATATCGAACTTTTCCTTGGAAATGCTGGAATTGCTATGCGTTCTCTGACAGCTGCTGTAACAGCCGCTGGAGGCAACTCAAGGTTCCTTCCCTCAACTCCTTCCAGC CTTTCAGCTTCTTH 253 18VAAGAAGCTGAAAGGCTGGAAGGAGTTGAGGGAAGGAACCTTGAGTTGCCTCCAGCGGCTGTTACAGCAGCTGTCAGTGAACGCATAGCGATTCCAGCATTTCCAAGGAAAAGTTCGATATCGTTCTTCCC CAGTTTACCGACCGH 254 19VCGGTCGGTAAACTGGGGAAGAACGATATCGAACTTTTCCTTGGAAATGCTGGAATCGCTATGCGTACTCTGACAGCTGCTGTAACAGCCGCTGGAGGCAACTCAAGGTTCCTTCCCTCAACTCCTTCCAGC CTTTCAGCTTCTTH 255 20VAAGAAGCTGAAAGGCTGGAAGGAGTTGAGGGAAGGAACCTTGAGTTGCCTCCAGCGGCTGTTACAGCAGCTGTCAGTGTACGCATAGCTATTCCAGCATTTCCAAGGAAAAGTTCGATATCGTTCTTCCC CAGTTTACCGACCGH 256 21VCGGTCGGTAAACTGGGGAAGAACGATATCGAACTTTTCCTTGGAAATGCTGGAATCGCTATGCGTGCGCTGACAGCTGCTGTAACAGCCGCTGGAGGCAACTCAAGGTTCCTTCCCTCAACTCCTTCCAGC CTTTCAGCTTCTTH 257 22VAAGAAGCTGAAAGGCTGGAAGGAGTTGAGGGAAGGAACCTTGAGTTGCCTCCAGCGGCTGTTACAGCAGCTGTCAGCGGACGCATAGCTATTCCAGCATTTCCAAGGAAAAGTTCGATATCGTTCTTCCC CAGTTTACCGACCGH 258 23VCGGTCGGTAAACTGGGGAAGAACGATATCGAACTTTTCCTTGGAAATGCTGGAATAGCTATGCGTGCTCTGACAGCTGCTGTAACAGCCGCTGGAGGCAACTCAAGGTTCCTTCCCTCAACTCCTTCCAGC CTTTCAGCTTCTTH 259 24VAAGAAGCTGAAAGGCTGGAAGGAGTTGAGGGAAGGAACCTTGAGTTGCCTCCAGCGGCTGTTACAGCAGCTGTCAGGGAACGCATAGCTGTTCCAGCATTTCCAAGGAAAAGTTCGATATCGTTCTTCCC CAGTTTACCGACCGH

Example 24: Precision Genome Editing Tools for Non-Transgenic CropBreeding

The following example demonstrates efficient and precise gene edits inArabidopsis thaliana protoplasts with exogenously introduced GRON alongwith an engineered nuclease, crispr-cas9. This genome editing technologyis also applied to agriculturally important crops such as Linumusitatissimum (flax) where, in protoplasts, precise edits in the EPSPSgenes provide a glyphosate tolerance trait; and subsequently regeneratedshoots exhibit those precise edits and the accompanying trait withoutthe need for selection.

Methods

Construction of CRISPS-Cas9 Plasmids

For construction of transient CRISPR-Cas9 expression plasmids, a higherplant codon-optimized SpCas9 gene containing a SV40 NLS at both the N-and C-terminal and a 3×FLAG tag on the N-terminal was synthesized as aseries of GeneArt® Strings™ (Life Technology, Carlsbad, Calif.), thencloned downstream of the mannopine synthase (MAS) promoter and upstreamof the pea ribulose bisphosphate carboxylase (rbcsE9) terminator byGibson's method²³ (FIG. 27 a ). Next, a sgRNA cassette consisting of achimeric gRNA, whose expression is driven by the Arabidopsis U6promoter, was synthesized as GeneArt® Strings™, then shuttled into theCas9 containing construct using Gibson's method forming pBCRISPR. Tospecify the chimeric sgRNA for the respective target sequence, pairs ofDNA oligonucleotides encoding the protospacers for BFP_sgRNA-1 andEPSPS_sgRNA-2 9 as shown in the following table were annealed togenerate short double stranded fragments with 4-bp overhangs).

ID 5′-3′ Off-1F GGAAGCAAACAGGTGACAGC Off-1R CGTATTTAGCCTCATCCAATGCOff-2F AAGGCTCCTCCAACTTCACC Off-2R TTCTCTGACTCTGATGGAGACC Off-3FCCCTTGGTGCAACATAAACC Off-3R GCGATGAATTTGAATTTTGACC Off-4FTTCGGGTTTAACGGGACAG Off-4R CGATTCCGGTAATTCACATTG Off-5FAAACCCTAGTGGCAGTTTCG Off-5R CGGTGGAAGCCCTGTTTAT BFPF-1TAAACGGCCACAAGTTCAGC BFPR-1 GGACGACGGCAACTACAAGACC LuEPF-1GCATAGCAGTGAGCAGAAGC LuEPR-1 AGAAGCTGAAAGGCTGGAAG CR-LuEPSPS2aGATTGCTGTTACAGCAGCTGTCAG CR-LuEPSPS2b AAACCTGACAGCTGCTGTAACAGC CR-BFP1aGATTGTCGTGACCACCTTCACCCA CR-BFP1b AAACTGGGTGAAGGTGGTCACGAC (SEQ ID NOS:260-277, respectively)

Figure references for these sequences are as follows: Off-1F throughBFPR-1-FIG. 27 d ; LuEPF-land LuEPR-1-FIG. 29 d ; CR-LuEPSPS2a andCR-LuEPSPS2b-FIG. 29 a ; CR-BFPa1 and CR-BFP1b-FIG. 27 a.

The fragments were ligated into BbsI digested pBCrispr to yieldCRISPR-Cas 9 constructs sgRNA (BFP)-1 and sgRNA (EPSPS)-2. Protospacersequences of sgRNA used are shown in the following table: (SEQ ID NOS:283 and 284)

FIG. ID Sequence 5′-3′ reference BFP_sgRNA-1 GTCGTGACCACCTTCACCCA FIG. 1EPSPS_sgRNA-2 GCTGTTACAGCAGCTGTCAG FIG. 3 G in red font altered in thesgRNA sequence to accommodate Pol III promoter

Gene Repair Oligonucleobases (GRONs)

All GRONs were purchased from Trilink Biotechnologies (San Diego,Calif.). A list of GRONS used are in the following table (SEQ ID NOS:285-289):

ID Target Sequence 5′-3′ Modification^(a) BFP/41 BFPGCTGAAGCACTGCACGCCGTAGGTAAACGTGGTCACGAGGGTGGG None BFP/41/3PS BFPG*C*T*GAAGCACTGCACGCCGTAGGTAAACGTGGTCACGAGGGT*G*G*G (*) = PS BP/101 BFPGTCGTGCTGCTTCATGTGGTCGGGGTAGCGGCTGAAGCACTGCACGCCGTACGTA NoneAACGTGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGG BFP/101/3PS BFPG*T*C*GTGCTGCTTCATGTGGTCGGGGTAGCGGCTGAAGCACTGCACGCCGTAC (*) = PSGTAAACGTGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGG*T*G*G EPSPS/144/Cy3 EPSPSVGCCAGCCATTTGACCSCTTCTTGCTATARCTTGAAAAGGAACCTTTACGACCTTAT V = Cy3; H= idC CGATACGCACGCGACTGTCGACGACATTGTCGGCGACCTCCGTTGAGTTCCARGGAAGGGAGTTGAGGAAGGTCGCAAAGTCGAAGAAH ^(a)PS - phosphorothioate bond; idC -reverse base; Cy3 - cyanine dye

Figure references for these sequences are as follows: BFP/41: FIGS. 27e, 28 a, 28 b ; BFP/41/3PS: FIG. 28 b ; BFP/101: FIG. 28 b ;BFP/101/3PF: FIG. 28 b ; EPSPS/144/Cy3: FIG. 29 .

Cell Culture and Protoplast Isolation

Arabidopsis

Surface-sterilized Arabidopsis seeds were germinated on solid t MSmedium (MS minerals and vitamins²⁴; ½ concentrated; 87.7 mM sucrose) at25° C. under a 12 h light/dark cycle. Roots from 2 to 3-week-oldseedlings were collected and maintained in MS liquid medium under lowlight at 25° C. Cultures were transferred to and maintained in MSAR1.1²⁵(½×MS salts with vitamins, Sucrose 87.7 mM, IAA, 11.4 μM, 2,4-D 4.6 μM)three weeks prior to protoplast isolation to induceroot-meristematic-tissue (RMT). RMT was cut into small segments andincubated in MSAR1.2 enzyme solution (MSAR1.1 containing 400 mMmannitol, 1.25% Cellulase RS, 0.25% Macerozyme R-10, 5 mM MES, 0.1% BSA)for 16 h in the dark with gentle shaking. The released protoplasts werecollected and passed consecutively through a sterile 100 μm and 35 μmfilter. The protoplast filtrate was added to 0.8 times the volume ofOptiprep™ Density Gradient Medium (Sigma) and mixed gently. A 60% W5(154 mM NaCl, 5 mM KCl, 125 mM CaCl₂.2H₂O, 5 mM glucose, 10 mM MES, pH5.8)/40% Optiprep solution followed by a 90% W5/10% Optiprep solutionwas slowly layered onto the filtrate/Optiprep solution to make agradient, which was centrifuged at 198 RCF for 10 min. The whiteprotoplast layer was collected and mixed with 2 times the volume of W5.Protoplasts were centrifuged at 44 RCF for 10 min and re-suspended in TMsolution (14.8 mM MgCl₂.6H₂O, 5 mM MES, 572 mM mannitol, pH 5.8) at adensity of 1×10⁷ cells/ml.

L. usitatissimum

Flax protoplasts were isolated from 3-week-old seedlings germinated invitro. Plant tissue was finely chopped with a scalpel, pre-plasmolyzedfor 1 h at room temperature in B-medium [B5 salts and vitamins²⁶, 4 mMCaCl₂, 0.1 M glucose, 0.3 M mannitol, 0.1 M glycine, 250 mg/l caseinhydrolysate, 10 mg/l L-cystein-HCL, 0.5% polyvinylpyrrolidone (MW10,000), 0.1% BSA, 1 mg/l BAP, 0.2 mg/l NAA, and 0.5 mg/l 2,4-D], andincubated in a cell wall digesting enzyme solution containing B-mediumsupplemented with 0.66% Cellulase YC and 0.16% Macerozyme R-10 on arotatory shaker (50 rpm) at 25° C. for 5 h. Released protoplasts weresieved and purified by density gradient centrifugation using Optipreplayers, counted with a hemocytometer, and kept stationary overnight inthe dark at a density of 0.5×10⁶ protoplasts/ml in B medium.

Protoplast Transfection

Arabidopsis

In a 96-well flat bottom plate, 2.5×10⁵ Arabidopsis protoplasts per wellwere transfected with either 2.5 pmol GRON alone, 2.5 pmol GRON plus 5μg CRISPR-Cas9 plasmid (BFP_sgRNA-1), or mock-treated using PEG mediateddelivery [270 mM mannitol, 67.5 mM Ca(NO₃)₂, 38.4% PEG 1500].Transfection occurred on ice for 10 minutes followed by a wash with 200μl of W5 solution. Finally, 85 μl of MSAP (MSAR1.1 containing 0.4 Mmannitol) was added and the cells cultured in low light conditions at25° C.

L. usitatissimum

After 18 h of culture, 1×10⁶ flax protoplasts were transfected with 200pmol of GRON along with 20 μg of CRISPR-Cas9 plasmid (EPSPS_sgRNA-2)using PEG mediated delivery. Treated protoplasts were incubated in thedark at 25° C. for up to 24 h in B medium, embedded in alginate beads²⁷at a density of 0.5×10⁶ protoplasts/ml alginate, and cultured in basalV-KM liquid medium 28 supplemented with 0.02 mg/l thidiazuron (TDZ) and0.002 mg/l NAA. EPSPS gene targeted sequence edits were assessed by NGSin gDNA extracted from pools of approximately 10,000 microcoloniesobtained from protoplasts 3 and 7 weeks after transfection. Microcalliwere then released from the alginate in 50 mM citrate buffer for 30 min,rinsed twice with V-KM medium, and plated on solidified regenerationmedium [MS salts²⁵, Morel and Wetmore vitamins²⁹ [0.001 mg/l biotin,0.01 mg/l nicotinic acid, 1 mg/l calcium pantothenate, 1 mg/lpyridoxine, 1 mg/l thiamine, 100 mg/l inositol), 3% sucrose, 0.02 mg/lthidiazuron (TDZ), 0.002 mg/l NAA, pH 5.8] at a density of 0.5 ml ofsettled cell volume/plate. Plated microcalli were incubated under a 16 hphotoperiod (270 mol·m⁻²·s⁻¹), at 25° C. After about 3 weeks, smallindividual calli (˜0.5 cm diameter) were split in two. One half was usedfor molecular screening, and the other half was kept in a 24-well plate,one callus per well, under the same conditions as for shootregeneration. Shoots began to develop from calli after about 6 weeks.Elongated shoots were micropropagated and rooted in MS medium (4.33 g/LMS salts mixture, 3% sucrose and 0.1% Morel and Wetmore vitamins, 0.3%Phytagel), and rooted plants were transferred to soil and hardened in agrowth chamber (Conviron, Winnipeg, MB) for 2-4 weeks until the plantswere well established.

Detection of Arabidopsis BFP to GFP Edits

Seventy-two hours after transfection, Arabidopsis protoplasts wereanalyzed by cytometry using the Attune® Acoustic Focusing cytometer(Applied Biosystems®) with excitation and detection of emission settingsas appropriate for GFP. Background level was based on PEG-treatedprotoplasts without DNA delivery.

Indel Analysis

Genomic DNA was extracted from treated protoplasts using the NucleoSpin®Plant II kit as per the manufacturer's recommendations (Machery-Nagel,Bethlehem, Pa.). Amplicons were generated with primers flanking theBFP_sgRNA-1 target region (BFPF-1 and BFPR-1) using Phusion® polymeraseand 100 ng of genomic DNA. The amplicons were purified and concentratedusing Qiaquick MinElute columns (Qiagen, Valencia, Calif.), then deepsequenced using a 2×250 bp MiSeq run (Illumina, San Diego, Calif.). Fordata analysis, fastq files for read 1 and read 2 were imported into CLCGenomics Workbench 7.0.4 (CLCBio, Boston, Mass.). Paired reads weremerged into a single sequence if their sequences overlapped. A sequencefor an amplicon was identified if it or its reverse and complementedsequence contained both forward and reverse primer sequences. Occurrenceof a unique sequence in a sample was recorded as its abundance. Percentindel or targeted edit was calculated by dividing the number ofsequences with the edit or indel by the total number of sequences, andthen multiplying by 100.

For flax samples, genomic DNA from microcolony or callus samples wasextracted using the EvoPURE plant DNA kit (Aline Biosciences, Woburn,Mass.) and screened by deep sequencing and allele-specific qPCR.Positive scoring PCR fragments were then TOPO-TA cloned into the pCR2.1vector (Invitrogen) per the manufacturer's protocol. Typically, clonedPCR fragments from 10-15 transformants were then TempliPhi sequenced (GEHealthcare Life Sciences) to confirm DNA sequence for each EPSPS allelefrom a single isolated callus. A similar PCR cloning and sequencingprocedure was used for DNA sequence confirmation in leaf samples ofregenerated shoots.

Off-Target Analysis

Potential off-target loci for BFP_sgRNA-1 in the Arabidopsis genome weredetermined using Cas-OFFinder. Five off-target sites based on sequenceidentities to bases 1-12 of the protospacer (seed sequence) werescreened for mutations. Arabidopsis protoplasts were transfected withCRISPR-Cas9 plasmid BFP_sgRNA-1 as described previously. After 72 h,gDNA was extracted and amplicons generated with Phusion polymerase (NEB)using primers that flank the potential off-target site (FIG. 27 d ). Theamplicons were then deep sequenced using a 2×250 bp MiSeq run. Thepercentage of indels was calculated by dividing the number of sequenceswith the edit or indel by the total number of sequences, and thenmultiplying by 100.

Molecular Screening of L. usitatissimum Plant Material

Genomic DNA from microcolony or microcallus samples was extracted usingthe EvoPURE plant DNA kit (Aline Biosciences, Woburn, Mass.) andscreened using allele-specific qPCR. Positively scoring PCR fragmentswere then TOPO-TA cloned into the pCR2.1 vector (Invitrogen) per themanufacturer's protocol. Typically, cloned PCR fragments from 10-15transformants were then TempliPhi sequenced (GE Healthcare LifeSciences) to confirm DNA sequence for each EPSPS allele from a singleisolated callus. A similar PCR cloning and sequencing procedure was usedfor DNA sequence confirmation of obtained shoots.

Herbicide Tolerance Tests

Glyphosate tolerance of calli and regenerated plants was assessed invitro and in the greenhouse, respectively. Individual calli were clonedby cutting and culturing smaller pieces in fresh regeneration medium toincrease callus mass. Calli derived from wild type leaf protoplasts wereused as negative control. Calli derived from subcultures of individualcallus lines were then pooled and broken up into 0.5-1 mm pieces byblending in liquid MS medium (4.33 g/L MS salts, 3% sucrose and 0.1%Morel and Wetmore vitamins) and 0.25 ml of settled callus pieces wasinoculated on regeneration medium containing 0, 0.125, 0.25, 0.5, 1.0 or2.0 mM glyphosate. Treatments were performed in triplicate, and theexperiments were repeated three times. Prior to spray tests, regeneratedplants were subjected to a hardening period in a growth chamber(Conviron, Winnipeg, MB) under a 16-h photoperiod with day and nighttemperatures 21° C. and 18° C. respectively. Hardened plants weretransferred to the greenhouse for glyphosate treatment. Wild typecontrol and EPSPS edited plants were sprayed with 10.5 mM or 21.0 mMglyphosate (Roundup Pro, Monsanto). Treatment rates were normalized to a75.7 ai/A (active ingredient per acre) spray volume to replicate fieldconditions. A mock treatment of surfactant only was included as acontrol. Plants were evaluated and photographed 6 days after theglyphosate treatment to determine herbicide tolerance. Statisticalanalysis

Statistical significance was determined using a Student's t-test withtwo-tailed distribution. P-values<0.05 were considered as significant.Data are shown as mean±SEM.

Discussion

In order to enhance GRON-mediated gene editing, the CRISPR-Cas9 systemwas used in this example. While exemplified in terms of the CRISPR-Cas9system, engineered nucleases can be programmed to target and cleavedouble-stranded DNA, and so would be expected to function equivalentlyin enhancing GRON-mediated editing. This would include nucleases such asmeganucleases, zinc finger nuclease (ZFN), and TAL effector nucleases(TALENs). In plants, DNA double-strand breaks (DSBs) are typicallyrepaired by the error-prone non-homologous end joining (NHEJ) DNA repairpathway, resulting in random deletions, insertions (indels) and/orsubstitutions at the site of repair. Precision genome editing relies onnuclease induced DSBs near the targeted locus to be repaired by homologydirected repair (HDR), a repair pathway that is more precise than NHEJdue to the requirement of a homologous DNA template-in most cases sisterchromatid. By harnessing the HDR pathway, it is possible to use GRONs inconjunction with engineered nucleases, to make scarless, precise customedits to targeted DNA.

The CRISPR-Cas9 system consists of two components: a Streptococcuspyogenes Cas9 nuclease (SpCas9) and a chimeric fusion of two RNAs (crRNAand tracrRNA) referred to as an engineered single guide RNA (sgRNA). ThesgRNA supports targeted nucleic acid specificity for Cas9 through basepairing of its first twenty 5′ bases with the DNA target, subsequentlyresulting in a site-specific DSB.

The efficacy of the CRISPR-Cas9 editing system in A. thalianaprotoplasts derived from a BFP transgenic model was used to demonstratethe efficacy of this system. In this model, aa stably integrated BFPgene can be converted to GFP by editing the codon encoding H66 (CAC) toY66 (TAC) (FIG. 27 a, b ). Activity of CRISPR-Cas9 plasmid, BFP_sgRNA-1targeting the BFP H66 locus was demonstrated by identifying indel scarsleft by NHEJ repair events. When protoplasts were treated withBFP_sgRNA-1, indels were detected at a frequency of 14.5% by deepsequencing, indicating a high efficiency of targeting (FIG. 27 c ).Notably, deletions outnumbered insertions with the majority of indelsfor either type being a single base pair (FIG. 27 c ). In protoplaststreated with Cas9 alone or mock-treated, indels were not detectedneighboring the H66 target site (data not shown).

Having established activity of BFP_sgRNA-1 in protoplasts from thismodel system, potential off-target activity of BFP_sgRNA-1 was examinedby searching the Arabidopsis genome using Cas-OFFinder for candidateoff-target sequences with high similarity to the BFP_sgRNA-1 targetsequence¹⁷. Using deep sequencing, five sites that, based on searches,contained the most homology to the BFP_sgRNA-1 target sequence wereexamined^(18, 19). Of the five sites tested, indel events were detectedat a very low frequency for only one locus, Off-1 (FIG. 27 d ). Whiledetectable, this level was 24-fold weaker when compared to the On-targetcontrol. It uis suspected that the activity observed at the Off-1 locusis based on homology of the sequence proximal to the PAM site where onlyone mismatch is present.

Next the effectiveness of GRON in combination with BFP_sgRNA-1 tofacilitate BFP to GFP precision gene editing was examined in the modelsystem. A marked improvement in BFP to GFP editing, as analyzed by flowcytometry, was identified after both GRON and BFP_sgRNA-1 (BFP/41) areintroduced concurrently when compared to GRON alone treatments (FIG. 27e ). Collectively, these results demonstrate that CRISPR-Cas9 plasmidBFP sgRNA-1 can actively disrupt the H66 locus of the BFP gene, andleave negligible off-target footprints. Further, when GRON is introducedwith BFP_sgRNA-1, the frequency of precise and scarless BFP to GFP editsincreases significantly.

When a GRON containing three adjacent PS modifications (BFP/41/3PS)combined with BFP_sgRNA-1 was used, more BFP to GFP precise scarlessedits were identified as compared to an unmodified GRON (BFP/41; FIG. 28a ). A similar result was found when testing a second GRON modificationcontaining a 5′ cyanine dye Cy3 and a 3′ iDc reverse base (data notshown). GRON lengths of 41 and 101 nucleobases (nb) with and without 3PSmodification were also tested in conjunction with BFP_sgRNA-1. Inmultiple experiments, the 101 nb GRON consistently exhibited increasedediting efficiency that was independent of 3PS modification whencompared to the shorter 41 nb GRON (FIG. 28 b ). Notably, for both GRONlengths tested, 3PS modification was superior to unmodified with respectto BFP to GFP editing. 201 nb length GRONs exhibit similar editingefficiencies to 101-nb GRONs (data not shown). Collectively, these datademonstrate that 3PS modification as well as GRON length can greatlyimprove the frequency of gene editing in A. thaliana protoplasts whencombined with the engineered nuclease, CRISPR-Cas9.

To extend the application of GRON and engineered nuclease mediated geneediting to other plant systems, two EPSPS(5′-enolpyruvylshikimate-3-phosphate synthase) loci in L. usitatissimumwere also targeted. The EPSPS genes encode an enzyme in the shikimatepathway that participates in the biosynthesis of the aromatic aminoacids phenylalanine, tyrosine and tryptophan. In plants, EPSPS is atarget for the herbicide, glyphosate, where it acts as a competitiveinhibitor of the binding site for phosphoenolpyruvate.

In an effort to improve targeting efficiency, a 144 nb Cy3 modified GRON(EPSPS/144/Cy3) and a CRISPR-Cas9 plasmid (EPSPS_sgRNA-2) was designedto target a conserved sequence in both EPSPS genes near two loci (T97Iand P101A) that, when edited, will render the EPSPS enzyme tolerant toglyphosate (FIG. 29 ). The 144 nb Cy3 modified GRON containing thetargeted changes together with EPSPS_sgRNA-2 was delivered intoprotoplasts, followed by measurement of gene editing and indel formationin 21-day-old microcolonies derived from the treated protoplasts by deepsequencing. Precise, scarless gene editing frequencies ranged between0.09 and 0.19%, and indels ranged between 19.2 and 19.8% in threeindependent experiments (Table 1). In all experiments, both EPSPS genesshowed a proportional number of edits and indels (data not shown),suggesting that GRON and EPSPS_sgRNA-2 are effective at editing bothgenes.

TABLE 1 Summary of L. usitatissimum CRISPR-cas9 experiments targetingEPSPS Deep sequencing of microcolonies^(a) Calli genotyping results^(c)Experiment Precise Calli Calli with ID edits (%)^(b) Indels (%) screenedprecise edits FC-1 0.19 19.8 5,167 8 (0.15%) FC-2 0.1 19.2 N/A FC-3 0.0919.6 N/A ^(a)gDNA was isolated from pools of ~10,000 microcolonies, thenused as template to amplify the target region ^(b)Sequences with T97I(ACA > ATA) and P101A (CCG > GCG) changes only; data combined for gene 1& gene 2 ^(c)Individual callus was screened first by allele-specificPCR, then confirmed by Sanger sequencing

Out of 5167 calli, 8 (0.15%) were found to harbor both T97I and P101Achanges in at least one of the EPSPS genes (Table 1). This result, aswell as the frequency of indels, correlated well with the initial deepsequencing data obtained from 21-day-old microcolonies. Shoots wereregenerated under non-selective conditions from the positive callusmaterial and successively confirmed the presence of the T97I and P101Aedits through DNA cloning and Sanger sequencing (FIG. 29 c ). These datashow that precise, targeted edits in the two endogenous EPSPS genes aswell as indel scars can be generated in L. usitatissimum protoplasts andsubsequently can be identified and maintained non-selectively in theprocess of shoot regeneration.

Calli that screened positive for precise edits were used to regeneratewhole plants under non-selective conditions-100% of which screenedpositive for the presence of the T97I and P101A edits in at least oneEPSPS gene through DNA cloning and Sanger sequencing (FIG. 29 b -6). Toidentify potential off-target mutations arising from treatment with byEPSPS_gRNA-2 in regenerated plants (Y23), we PCR amplified regionscontaining sequence similarity to the EPSPS_sgRNA-2 protospacer andmeasured for NHEJ scars by deep sequencing. No mutations were identifiedin any of these potential off-target sites (SEQ ID NOS: 290-298):

Off-Target # of Mutations ID Scaffold or Locus ID^(a) Position^(b) OffTarget Sequence^(c) Mismatches detected^(d) Off-1 C7813595 197-219ccgGTTACAGCAGCaGTCgGCGG 5 − Off-2 Lus10030959.g 243476-243460ccgGTTACAGCAGCaGTCgGCGG 5 − Off-3 Scaffold 155 681644-681624tcaaaagCtGCAGCTaTCAGTGG 9 − Off-4 Lus10036882.g 1067934-1067911tcaaaatCtGCAGCTGTCAGTGG 8 − Off-5 Scaffold 107 1077588-1077568tcaaaatCtGCgGCTGTCAGTGG 9 − Off-6 Scaffold 743 195079-195059tcaaaatCtGCgGCTGTCAGTGG 9 − Off-7 Scaffold 208 238604-238626aaggacACAGCAGCTGTCgGTGG 7 − Off-8 Scaffold 2252 38795-38773accaaacgAGCAGCTGTCAGAGG 8 − On Lus10000788.g 19227-19249GCTGTTACAGCAGCTGTCAGCGG 0 + ^(a)Scaffold or locus ID from Phytozyme 10.2^(b)Protospacer position within scaffold ^(c)Red lowercase bases aremismatches to the EPSPS_sgRNA protospacer ^(d)Mutations determined bysequencing, On-target mutations are T97I and P101A

To determine the glyphosate tolerance afforded by the T97I and P101Amutations, we challenged callus line (Y23) that was identified as beingheterozygous for the T97I and P101A edits in EPSPS gene 2, as well aswhole plants regenerated from these calli with glyphosate. The freshweight of calli harboring the T97I and P101A edits was significantlyhigher (p<0.01) than that of wild type calli at all glyphosateconcentrations tested (FIG. 30 a, b). Wild type and edited whole plantswere grown in soil under greenhouse conditions, sprayed with a solutioncontaining either 10.5 or 21.0 mM glyphosate and evaluated 6 days aftertreatment. Wild type plants exhibited a wilted and necrotic phenotypetypical of glyphosate toxicity at both application rates, whereas plantsharboring the edited EPSPS gene exhibited minimal phenotypic change(FIG. 30 c ). This result implies that a single edited EPSPS geneprovides a level of tolerance much greater than that observed in thecontrol plants at two rates of glyphosate. Taken together, these datademonstrate that RTDS combined with CRISPR-Cas9 can result in precise,targeted edits at sufficient frequency such that these edits as well asindel scars can be detected by molecular screening and maintained undernon-selective culture conditions and efficiently regenerated intoplants.

Subsequent to this analysis, Oryza sativa calli with precise edits in agene imparting herbicide resistance have also been obtained using GRONin combination with CRISPR-Cas9. Further, reproducible edits in a BFP toGFP transgene using GRON combined with TALENs in A. thaliana and GRONcombined with CRISPR-Cas9 in Brassica napus have been obtained.

REFERENCES

-   1. Feng, Z. et al. Efficient genome editing in plants using a    CRISPR/Cas system. Cell Res. 23, 1229-32 (2013).-   2. Voytas, D. F. Plant genome engineering with sequence-specific    nucleases. Annu. Rev. Plant Biol. 64, 327-50 (2013).-   3. Jiang, W. et al. Demonstration of CRISPR/Cas9/sgRNA-mediated    targeted gene modification in Arabidopsis, tobacco, sorghum and    rice. Nucleic Acids Res. 41, e188 (2013).-   4. Li, J.-F. et al. Multiplex and homologous recombination-mediated    genome editing in Arabidopsis and Nicotiana benthamiana using guide    RNA and Cas9. Nat. Biotechnol. 31, 688-91 (2013).-   5. Mao, Y. et al. Application of the CRISPR-Cas system for efficient    genome engineering in plants. Mol. Plant 6, 2008-2011 (2013).-   6. Miao, J. et al. Targeted mutagenesis in rice using CRISPR-Cas    system. Cell Res. 23, 1233-6 (2013).-   7. Nekrasov, V. et al. Targeted mutagenesis in the model plant    Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nat.    Biotechnol. 31, 691-3 (2013).-   8. Shan, Q. et al. Targeted genome modification of crop plants using    a CRISPR-Cas system. Nat. Biotechnol. 31, 686-8 (2013).-   9. Ran, F. A. et al. Genome engineering using the CRISPR-Cas9    system. Nat. Protoc. 8, 2281-308 (2013).-   10. Inui, M. et al. Rapid generation of mouse models with defined    point mutations by the CRISPR/Cas9 system. Sci. Rep. 4, 5396 (2014).-   11. Wu, Y. et al. Correction of a genetic disease in mouse via use    of CRISPR-Cas 9.Cell Stem Cell 13, 659-62 (2013).-   12. Xue, W. et al. CRISPR-mediated direct mutation of cancer genes    in the mouse liver. Nature 514, 380-384 (2014).-   13. Zhao, P. et al. Oligonucleotide-based targeted gene editing    in C. elegans via the CRISPR/Cas9 system. Cell Res. 24, 247-50    (2014).-   14. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease    in adaptive bacterial immunity. Science 337, 816-21 (2012).-   15. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas    systems. Science 339, 819-23 (2013).-   16. Symington, L. S. & Gautier, J. Double-strand break end resection    and repair pathway choice. Annu. Rev. Genet. 45, 247-71 (2011).-   17. Bae, S. et al. Cas-OFFinder: a fast and versatile algorithum    that searches for potenial off-target sites of Cas9 RNA-guided    endonucleases. Bioinformatics. 30, 1473-1475 (2014).-   18. Jiang, W. et al. RNA-guided editing of bacterial genomes using    CRISPR-Cas systems. Nat. Biotechnol. 31, 233-239 (2013)-   19. Hsu, P. et al. DNA targeting specificity of RNA-guided Cas9    nucleases. Nat Biotechnol. 31, 827-832 (2013)-   20. Papaioannou, I. et al. Use of internally nuclease-protected    single-strand DNA oligonucleotides and silencing of the mismatch    repair protein, MSH2, enhances the replication of corrected cells    following gene editing. J. Gene Med. 11, 267-74 (2009).-   21. Schönbrunn, E. et al. Interaction of the herbicide glyphosate    with its target enzyme 5-enolpyruvylshikimate 3-phosphate synthase    in atomic detail. Proc. Natl. Acad. Sci. U.S.A. 98, 1376-80 (2001).-   22. Gocal, G., Knuth, M., Beetham, P. Generic EPSPS mutants. U.S.    Pat. No. 8,268,622. Filled Jan. 10, 2007, Issued Sep. 18, 2012.-   23. Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to    several hundred kilobases. Nat. Methods 6, 343-5 (2009).-   24. Murashige, T. & Skoog, F. A revised medium for rapid growth and    bio-assays with tobacco tissue cultures. Physiol Plant 15, 473-497    (1962).-   25. Mathur, J. & Koncz, C. A simple method for isolation, liquid    culture, transformation and regeneration of Arabidopsis thaliana    protoplasts. Plant Cell Rep. 10, 221-226 (1995).-   26. Gamborg, O. L. et al. Nutrient requirements of suspension    cultures of soybean root cells. Exp Cell Res 50, 151-8 (1968).-   27. Roger, D. et al. Immobilization of flax protoplasts in agarose    and alginate beads. Plant Physiol. 112, 1191-9 (1996).-   28. Binding, H & Nehls, R. Regeneration of isolated protoplasts to    plants in Solanum dulcamara L. Z. Pflanzenphysiol. 85, 279-280    (1977).-   29. Morel, G. & Wetmore, R. Fern callus tissue culture. Am J Bot.    38:141-143 (1951).-   30. Morlan, J. et al. Mutation detection by Real-Time PCR: A simple,    robust and highly selective method. PLoS ONE 4(2).

Example 24. Breakers and GRONs in Flax

Design and construction of TALEN expression constructs BT-1 and LuET-1was based on rules as described in Cermek et al. (2011). The targetsequence was selected based on the gene editing site and the repeatvariable i-residue (RVD) following the rules that NG, HD, NI, and NNrecognize T, C, A, and G, respectively. The assembly of TAL effectordomain linked to the heterodimeric FokI domains was completed through acommercial service (GeneArt; Life Technologies). TALEN monomers werecloned between the MAS promoter and the rbcE9 terminator using Gibson'smethod (Gibson et al., 2009) and expressed as a coupled unit (FIGS. 33 aand 36 a ). All GRONs were purchased from Trilink BioTechnologies (SanDiego, Calif.).

GRONs used in this study (SEQ ID NOS: 299-304):

Name Sequence(5′ to 3′) Chemistry BFP/41G*C*T*GAAGCACTGCACGCCGTAGGTGAAGGTGGTCACGA*G*G*G (*) = 3PS BFP/41/NTG*C*T*GAAGCACTGCACGCCGTGGGTGAAGGTGGTCACGA*G*G*G (*) = 3PS BFP/101G*T*C*GTGCTGCTTCATGTGGTCGGGGTAGCGGCTGAAGCACTGCACGCCGTAGGTGAAGGTGGTCACGAGGGTGGGCCA(*) = 3PS GGGCACGGGCAGCTTGCCGG*T*G*G BFP/201A*A*G*ATGGTGCGCTCCTGGACGTAGCCTTCGGGCATGGCGGACTTGAAGAAGTCGTGCTGCTTCATGTGGTCGGGGTAGC(*) = 3PSGGCTGAAGCACTGCACGCCGTAGGTGAAGGTGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGATGAACTTCAGGGTCAGCTTGCCGTAGGTGGCATCGCCCTCG*C*C EPSPS/144*VCGGTCGGTAAACTGGCGAAGAACGATATTGAACTTTTCCTTGGAAATGCTGGAATAGCTATGCGTGCGCTGACAGCTGCTGV = CY3;TAACAGCCGCTGGAGGCAACTCAAGGTCCCTTCCCTCAACTCCTTCCAGCCTTTCAGCTTCTTH M= 3′DMT and dC CPGVCGGTCGGTAAACTGGCGAAGAACGATATTGAACTTTTCCTTGGAAATGCTGGAATAGCTATGCGTGCGCTGACAGCTGCTGTAACAGCCGCTGGAGGCAACTCAAGGTTCCTTCCCTCAACTCCTTCCAGCCTTTCAGCTTCTTH*EPSPS/144 consists of an equimolar mixture of two oligos, eachcontaining sequences specific for SNPs for EPSPS gene 1 and 2. Bothcontain the T97I and P101A edits.

Figure references for these sequences are as follows: BFP/41: FIGS. 31b, 32 a, 35 b ; BFP/41/NT: FIG. 32 a ; BFP/101: FIGS. 31 b, 35 b ;BFP/201: FIGS. 31 b, 35 b ; EPSPS/144: FIG. 36 b.

TALE binding doiman sequence (SEQ ID NOS: 305-308):

TALEN ID Sequence (5′ to 3′) BT-1 Left arm: TGGTCGGGGTAGCGGCTGA Rightarm: TCGTGACCACCTTCACCCA LuET-1 Left arm: TGGAACAGCTATGCGTCCG Right arm:TGAGTTGCCTCCAGCGGCT

Figure references for these sequences are as follows: BT-1: FIGS. 33 aand b; LuET-1: FIGS. 36 a and b.

Primers used in this study (SEQ ID NOS: 309-312):

ID Sequence 5′-3′ BFPF-1 TAAACGGCCACAAGTTCAGC BFPR-1GGACGACGGCAACTACAAGACC LuEPF-1 GCATAGCAGTGAGCAGAAGC LuEPR-1AGAAGCTGAAAGGCTGGAAG

Figure references for these sequences are as follows: BFPF-1: FIGS. 33 dand e, 34a and b; BFPR-1: FIGS. FIGS. 33 d and e, 34a and b; LuEPF-1:FIGS. 37 a, b, c, and d, 38a; LuEPR-1: FIGS. 37 a, b, c, and d, 38b.

Arabidopsis:

Surface-sterilized Arabidopsis seeds were germinated on solid ½ MSmedium (MS medium containing half the concentration of minerals andvitamins; 87.7 mM sucrose; Murashige and Skoog, 1962) at 25° C. under a12 h light/dark cycle. Roots from 2 to 3-week-old seedlings werecollected and maintained in MS liquid medium in the dark at 25° C.Cultures were transferred to and maintained in MSAR1.1 (MSAR with 11.4μM IAA, 4.6 μM 2,4-D; Mathur and Koncz, 1995) three weeks prior toprotoplast isolation to induce root-meristematic-tissue (RMT). RMT wascut into small segments and incubated in MSAR1.2 enzyme solution(MSAR1.1 containing 0.4 M mannitol, 1.25% cellulase RS, 0.25% macerozymeR-10, 5 mM MES, 0.1% BSA) for 16 h in the dark with gentle shaking. Thereleased protoplasts were collected and passed consecutively through asterile 100 μm and 35 μm filter. The protoplast filtrate was purified bydensity centrifugation. The protoplast layer was collected and mixedwith 2 times the volume of W5 (154 mM NaCl, 5 mM KCl, 125 mM CaCl₂.2H₂O,5 mM glucose, 10 mM MES, pH 5.8; Menczel et al., 1981). Protoplasts werecentrifuged at 44 RCF for 10 min and re-suspended in TM solution (14.8mM MgCl₂.6H₂O, 5 mM MES, 572 mM mannitol, pH 5.8) at a density of 1×10⁶cells/ml. For experiments with phleomycin (InvivoGen, San Diego,Calif.), protoplasts were kept in TM adjusted to pH 7.0 for 90 min onice before transfection. For antibiotic concentrations see FIG. 31 a.

In a 96-well flat bottom plate, 2.5×10⁵ Arabidopsis protoplasts per wellwere transfected using PEG mediated delivery [270 mM mannitol, 67.5 mMCa(NO₃)₂, 38.4% PEG 1500] with either 1.2 nmol GRON alone, 93 pmol GRONplus 7.5 μg BFP TALEN plasmid (BT-1), or without DNA. Transfectionoccurred on ice for 10 minutes followed by a wash with 200 μl of W5solution. Finally, 85 μl of MSAP (MSAR1.1 containing 0.4 M mannitol) wasadded and the cells cultured in dark conditions at 25° C.

L. usitatissimum:

Flax protoplasts were isolated from 3-week-old seedlings germinated invitro. Plant tissue was chopped with a scalpel, pre-plasmolyzed for 1 hat room temperature in B-medium [B5 salts and vitamins, 4 mM CaCl₂, 0.1M glucose, 0.3 M mannitol, 0.1 M glycine, 250 mg/l casein hydrolysate,10 mg/l L-cysteine-HCl, 0.5% polyvinylpyrrolidone (MW 10,000), 0.1% BSA,1 mg/l BAP, 0.2 mg/l NAA, and 0.5 mg/l 2,4-D; Gamborg et al., 1968], andincubated in a cell wall digesting enzyme solution containing B-mediumsupplemented with 0.66% cellulase YC and 0.16% macerozyme R-10 on arotatory shaker (50 rpm) at 25° C. for 5 h. Released protoplasts weresieved and purified by density gradient centrifugation and keptstationary overnight in the dark at a density of 0.5×10⁶ protoplasts/mlin B medium.

After 18 h of culture, 1×10⁶ flax protoplasts were transfected with 200pmol of GRON along with 20 μg of EPSPS TALEN plasmid (LuET-1) usingPEG-mediated delivery. Treated protoplasts were incubated in the dark at25° C. for up to 24 h in B medium, embedded in alginate beads (Roger etal., 1996) at a density of 0.5×10⁶ protoplasts/ml alginate, and culturedin basal V-KM liquid medium (Binding and Nehls, 1977) supplemented with0.02 mg/l thidiazuron (TDZ), and 0.002 mg/l NAA. EPSPS gene targetedsequence edits were assessed by NGS in gDNA extracted from approximately50,000 cells one week after transfection.

Detection of Arabidopsis BFP to GFP edits: Seventy-two hours aftertransfection, Arabidopsis protoplasts were analyzed by cytometry usingthe Attune® Acoustic Focusing cytometer (Life Technologies, Carlsbad,Calif.) with excitation and detection of emission settings asappropriate for GFP. Background level was based on PEG-treatedprotoplasts without DNA delivery. For antibiotic experiments,protoplasts treated with phleomycin prior to transfection were analyzedby cytometry 24 h after transfection.

Indel analysis: Genomic DNA was extracted from either Arabidopsis orflax treated protoplasts using the NucleoSpin® Plant II kit as per themanufacturer's recommendations (Machery-Nagel, Bethlehem, Pa.).Amplicons were generated with primers flanking the BT-1 or LuET-1 TALENtarget region (FIG. 38 ) using Phusion® polymerase and 100 ng of genomicDNA. The amplicons were purified and concentrated using QiaquickMinElute columns (Qiagen, Valencia, Calif.), then deep sequenced using a2×250 bp MiSeq run (Illumina, San Diego, Calif.). For data analysisFASTQ files for read 1 and read 2 were imported into CLC GenomicsWorkbench 7.0.4 (CLCBio, Boston, Mass.). Paired reads were merged into asingle sequence if their sequences overlapped. A sequence for anamplicon was identified if it or its reverse and complemented sequencecontained both forward and reverse primer sequences. Occurrence of aunique sequence in a sample was recorded as its abundance. Percent indelor targeted edit was calculated by dividing the number of sequences withthe edit or indel by the total number of sequences, and then multiplyingby 100.

Statistical Analysis

Statistical significance was determined using a Student's t-test withtwo-tailed distribution. P-values <0.05 were considered as significant.Data are shown as mean and SEM.

Results and Discussion

RTDS Technology Applied to Convert BFP to GFP in Arabidopsis

Rapid Trait Development System (RTDS™) is an advanced Oligo-DirectedMutagenesis (ODM) technology that uses the natural or inherent DNArepair system to enable editing of genes in a target specific manner. Ineukaryotic cells, the GRON enters the cell by crossing the cell membraneand subsequently traverses to the nucleus, where it locates and bindsselectively and specifically to the target sequence, resulting insequence specific change(s) in the target gene. Nucleases and otherdegrading enzymes in the cells break down the GRON after the target genehas been edited (FIG. 31 a ). To demonstrate the effectiveness of RTDSin Arabidopsis, protoplasts derived from BFP transgenic lines (in whicha stably integrated BFP gene can be converted to GFP by editing thecodon encoding H66 (CAC) to Y66 (TAC) (FIG. 31 b )) were used. Usingthis system, gene editing may be determined based on a cells' greenfluorescence by flow cytometry. Protoplasts from this line were treatedfor 90 min with 0, 250 or 1000 μg/mL of the glycopeptide antibioticphleomycin. We then introduced either ssODN BFP/41 or BFP/41/NT(BFP/41/NT serves as a negative control and does not contain the C→Tedit to convert BFP to GFP, and monitored GFP fluorescence by cytometry24 h after delivery. BFP/41 along with phleomycin pre-treatment resultedin a dose-dependent increase in the number of GFP positive cells (FIG.32 ). These results provide evidence that ssODNs can enhance thefrequency and precision of non-specific DSB reagents, such asphleomycin-based genome editing in Arabidopsis protoplasts.

RTDS Technology Combined with a Glycopeptide Antibiotic to Convert BFPto GFP in Arabidopsis

It was previously reported that mammalian cells exposed to a low dose ofglycopeptide antibiotic exhibited enhanced targeted gene editing whencombined with oligonucleotides harboring the desired base change (Suzukiet al., 2003). These reagents bind and intercalate DNA, destroying theintegrity of the double helix and resulting in a DNA double strandbreak. It is hypothesized that these antibiotics enhance ODM byelevating the expression/activity of DNA repair genes, causing doublestrand breaks near the target site or a combination of both. To test theeffect of low-dose glycopeptide antibiotic treatment in combination withGRON, Arabidopsis protoplasts were incubated for 90 min with 10, 250 and1000 μg/mL of the glycopeptide antibiotic phleomycin, then either GRONBFP/41 or the negative control GRON BFP/41/NT that does not contain theC>T edit was introduced and the protoplasts monitored GFP fluorescenceafter 24 h by cytometry. In treating protoplasts with lowestconcentration of phleomycin (10 μg/mL), no improvement in the number ofGFP positive cells when compared to the BFP/41/NT control was detected,despite using a concentration of antibiotic that is significantly higherthan that used by Suzuki et al. (2003; data not shown). However at thehigher 250 and 1000 μg/mL concentration, phleomycin pre-treatmentresulted in a dose-dependent increase in GFP positive cells, reaching aconversion frequency of 0.14% at 1000 μg/mL (FIG. 32 a ). FIG. 32 bshows edited Arabidopsis protoplasts 5 d after GRON delivery andtreatment with glycopeptide antibiotics.

It is possible that these higher levels of phleomycin treatment arerequired in Arabidopsis as compared with mammalian cells due to lowerpermeability of the plant cell membrane to the antibiotic. For examplein human fibroblasts, Sidik and Smerdon, (1990) found that pre-treatmentwith the membrane permeabilizer, lysophosphatidylcholine, prior totreating with bleomycin, significantly increased the amount of DNArepair synthesis when compared to cells not treated with thepermeabilizer. The authors attributed this effect to lower amounts ofbleomycin entering the cells. While it is unclear whether Arabidopsiscell membranes have a lower permeability to phleomycin, these results dodemonstrate that pre-treatment with high levels of phleomycinsignificantly increased GRON-mediated gene editing. It is uncertainwhether this positive effect on gene editing is based on increaseddouble strand DNA breaks near the target site, up-regulation of DNArepair pathways or a combination of both. There is potential forphleomycin to introduce other non-targeted changes in the DNA as aresult of imprecise NHEJ repair events due to its non-discriminatenature, however; such random mutations could be eliminated throughfollow-on plant breeding. These mutations are similar to the mutationsinduced using chemical mutagenesis as part of traditional breedingprograms. As such, the use of phleomycin as a method for improvingRTDS-based precise gene edits is notable and implies that generation ofDNA double strand breaks can be an important factor for improvingconversion efficiencies in plants.

RTDS Technology Combined with TALENs to Convert BFP to GFP inArabidopsis

A TALEN (BT-1) that will target and cleave just downstream of the C>Tedit required to convert BFP to GFP in our Arabidopsis BFP transgenicline (FIGS. 32 a and b ) was used in the following experiment. The BT-1TALEN pair recognizes two 17 bp sequences separated by a 12 bp spacerregion and was designed according to the architecture specificationsoutlined in Cermek et al. (2011). The target site (C>T) is within theleft TALEN arm and thus will be upstream from the predicted cleavagesite. Transient BT-1 protein expression was established in Arabidopsisprotoplasts by using a Western blot (FIG. 33 c ). Next, to evaluate thecleavage efficiency of BT-1, a genomic DNA from BT-1 transfectedprotoplasts was isolated 72 h after TALEN introduction and then theregion surrounding the TALEN binding sites was PCR amplified (FIG. 38 )using the primers listed above. The resulting amplicons were assessedfor indel scars or substitutions resulting from imprecise NHEJ eventsnear the TALEN cleavage site by deep sequencing. Protoplasts treatedwith BT-1 showed indel mutations at a frequency of 2.9%, andsubstitutions at 5.1% (FIG. 34 a ). Deletions were mostly ≤20 bp andsignificantly outnumbered insertions (FIG. 34 b ). The distribution ofindels ≤20 bp with respect to length in bp is shown in FIGS. 34 c and d.

After establishing the activity of BT-1 in targeting our BFP transgene,the combinatorial effect of GRON combined with BT-1 TALEN to mediate BFPto GFP conversion was tested. BT-1 TALEN was introduced, along withGRONs BFP/41, BFP/101 or BFP/201, that contain the nucleotide changeencoding a H66Y amino acid substitution, into Arabidopsis protoplasts.BFP/101 and BFP/201 also contain 5 silent mutations that would deterunwanted TALEN activity on a corrected GFP gene (FIG. 35 a ). GFPfluorescence was measured by flow cytometry 72 h after delivery of GRONsand TALENs. Protoplasts treated with both GRON and TALEN showedsignificantly more GFP fluorescing cells (25 to 45-fold) compared totreatments with either GRON alone (FIG. 31 c ) or mock treatments (FIG.35 b ). This finding is similar to that of Strouse et al. (2014), whereit was reported that single stranded oligos combined with TALENssignificantly increased gene editing rates in mammalian cells. Acomparable GRON length dependent positive effect was observed withrespect to gene editing in the combined GRON and TALEN treatments (FIG.35 b ). Taken together, these results demonstrate that employingRTDS-mediated gene editing with a targeted DNA double strand breaker(TALEN) in a combinatorial approach significantly improves the frequencyof BFP to GFP editing in a GRON length dependent manner in Arabidopsisprotoplasts.

Next, to determine if ssODNs can also positively influence genomeediting outcomes induced by DSB reagents that make more target-specificcuts, a TALEN expression construct (BT-1) that will target and make aDSB just downstream of the C→T edit required to convert BFP to GFP inour Arabidopsis transgenic line (FIG. 33C) was employed. The BT-1 TALENconstruct consists of two arms, both having a TAL effector-like DNAbinding domain (TALE) linked to a catalytic DNA nuclease domain of FokI.The TALE domains guide the TALEN arms to specific sites of DNA allowingfor dimerization of the FokI endonucleases and subsequent generation ofa targeted DNA double strand break in the spacer region between the twobinding sites (Cermak et al., 2011). Each BT-1 TALE recognizes a 19 bpsequence separated by a 14 bp spacer and is comprised of the truncatedN152/C+63 architecture (Miller et al., 2011).

BT-1 activity at the targeted site on the BFP transgene by measuringimprecise NHEJ repair events occurring in the spacer region was examinedas follows. Total genomic DNA was extracted from treated protoplasts 72hafter introduction of BT-1 and the target region amplified by PCR. PCRamplicons were then deep sequenced to a depth of >500,000 reads(Supplemental FIG. S1A and Table S3). Analysis showed the frequency ofdeletions and insertions averaged 2.6 and 0.3%, respectively (FIG. 37A).Deletions were primarily ≤20 bp while insertions were more equallydistributed with respect to size (FIG. 37B). After establishing thetargeting activity of BT-1 on the BFP transgene, we next tested theeffect of combining ssODNs with BT-1 TALEN to convert BFP to GFP. Forthese experiments, we examined three different length ssODNs (BFP/41,BFP/101 or BFP/201, each independently delivered with or without BT-1plasmid into Arabidopsis protoplasts. The resulting BFP to GFP editingwas then quantified by cytometry 72 h after delivery. Protoplaststreated with both ssODNs and BT-1 TALEN exhibited 25- to 45-fold moregreen fluorescing cells than treatment with ssODN alone and more than125-fold when TALENs are used alone (FIG. 37C). Notably, ssODN lengthhad a positive effect on the frequency of BFP to GFP edits, whethercombined with BT-1 TALEN or used alone. Taken together, these data showthat when ssODNs are combined with a target-specific DSB reagent, thefrequency of precise genome edits is increased by ˜10-fold over thatobserved with a non-specific DSB reagent.

ssODNs Combined with CRISPR/Cas9

Combining ssODNs with TALENs resulted in a significant improvement inthe frequency of genome edits over using TALENs or ssODNs alone.However, considering the complexity of re-engineering TALEN proteins foreach new DNA target, we asked if the more easily designed andconstructed engineered nuclease CRISPR/Cas9 could also show enhancedgenome edit frequency when supplied with ssODNs. The CRISPR/Cas9 systemconsists of a Streptococcus pyogenes Cas9 nuclease and a chimeric fusionof two RNAs (crRNA and tracrRNA) referred to as an engineered singleguide RNA (sgRNA). The sgRNA supports targeted nucleic acid specificityfor Cas9 through base pairing of its first twenty 5′ bases with the DNAtarget, resulting in a site-specific DSB (Cong et al., 2013). Incontrast with TALENs, changing the target specificity of the CRISPR/Cas9protein complex does not require extensive protein engineering but onlyminimal manipulation of the sgRNA. The CRISPR/Cas9 expression plasmid,BC-1 (FIG. 34A), was designed to target near locus H66 of the BFP genein our transgenic model (FIG. 34B) as shown in the following table (SEQID NOS: 313 and 314):

CRISPR/Cas9 ID Sequence 5′-3′ BC-1 GTCGTGACCACCTTCACCCA EC-2GCTGTTACAGCAGCTGTCAG G in undelined font altered in the sgRNA sequenceto accommodate Pol III promoter

Following a similar experimental methodology as in our TALENs work, theability of BC-1 to target and cleave the BFP gene was determined bymeasuring the frequency of imprecise NHEJ repair events found upstreamof the PAM sequence. In protoplasts treated with BC-1, we detecteddeletions and insertions at a frequency of 3.7 and 2.4%, respectivelyusing deep amplicon sequencing (FIG. 35A). The most represented indelfor either insertions or deletions was a single base pair (Data notshown). Notably, when compared to similar experiments with BT-1 (TALEN),the BFP transgene targeting efficiency of BC-1 (CRISPR/Cas9) was nearlythree times higher (FIG. 35B). Having established the on-target activityof BC-1, potential off-target cleavage eas tested by searching theArabidopsis genome for sequences with high similarity to the BC-1 targetsequence using Cas-OFFinder (Bae et al., 2014). Five potentialoff-target sites that, based on searches, exhibited the most homology tothe BC-1 target sequence (Hsu et al., 2013) were examined. Arabidopsisprotoplasts were treated with BC-1 for 72 h, after which amplicons weregenerated using primers that flank each of the five potential off-targetsites:

Primers used in this study ID Sequence 5′-3′ Species SEQ ID NO BFPF-1TAAACGGCCACAAGTTCAGC Arabidopsis 55 BFPR-1 GGACGACGGCAACTACAAGACCArabidopsis 54 LuEPF-1 GCATAGCAGTGAGCAGAAGC Flax 56 LuEPR-1AGAAGCTGAAAGGCTGGAAG Flax 57 Off-1FA GGAAGCAAACAGGTGACAGC Arabidopsis260 Off-1RA CGTATTTAGCCTCATCCAATGC Arabidopsis 261 Off-2FAAAGGCTCCTCCAACTTCACC Arabidopsis 262 Off-2RA TTCTCTGACTCTGATGGAGACCArabidopsis 263 Off-3FA CCCTTGGTGCAACATAAACC Arabidopsis 264 Off-3RAGCGATGAATTTGAATTTTGACC Arabidopsis 265 Off-4FA TTCGGGTTTAACGGGACAGArabidopsis 266 Off-4RA CGATTCCGGTAATTCACATTG Arabidopsis 267 Off-5FAAAACCCTAGTGGCAGTTTCG Arabidopsis 268 Off-5RA CGGTGGAAGCCCTGTTTATArabidopsis 269 Off-1FF CAAGGCTAATTAGACTTAGATGATGTGG Flax 315 Off-1RFGGTGCACCGCC Flax 316 Off-2FF CAAGGCTAATTAGACTTAGATGATGTGG Flax 315Off-2RF GGTGCACCGCC Flax 316 Off-3FF GCCATCATCGCCCTTTAAGC Flax 317Off-3RF TGGTGTTTTGCTCTGTGAACG Flax 318 Off-4FF GCCATCATCGCCCTTTAAGC Flax317 Off-4RF TGGTGTTTTGCTCTGTGAACG Flax 318 Off-5FF GCCATCATCGCCCTTTAAGCFlax 317 Off-5RF TGGTGTTTTGCTCTGTGAACG Flax 318 Off-6FFGCCATCATCGCCCTTTAAGC Flax 317 Off-6RF TGGTGTTTTGCTCTGTGAACG Flax 318Off-7FF GAAAGAAGGCACTCTCAGAACATAC Flax 319 Off-7RFTGAATTTTGCTATCCTCTTCCCAATTTG Flax 320 Off-8FF CGTACGTTGTCAAGAAGTGACCFlax 321 Off-8RF ACCAAGACGGTAGTGGATGTC Flax 322

These amplicons were then analyzed for NHEJ mutations by amplicon deepsequencing:

BC-1 off-targets (SEQ ID NOS: 323-328, respectively) Off-targetArabidopsis # of ID Chrom # Position Off-target sequence mismatches GeneOff-1 chr1 13067189 gTtGTtgCCACCTTCAaCCAAGG 5 intergenic Off-2 chr219145890 CaCGTccCCACCaTCtCCCAAGG 5 uncharacterized protein Off-3 chr517191806 tTCaTcACCAgCTTCACCaATGG 5 uncharacterized protein Off-4 chr4 4803635 tTCGTGctCACCTTCACggATGG 5 Cellulose-synthase like Off-5 chr414554739 CTCGaacCCACCTTCAgCaAAGG 5 polyamine oxidase 5 On NA NACTCGTGACCACCTTCACCCACGG NA BFP transgene

Of the five sites tested, only Off-1 showed mutations near the predictedcleavage site (FIG. 35C). While detectable, this level is ˜13-fold lessthan the On-target control. This weak activity at Off-1 is likely basedon homology of the sequence proximal to the PAM site where only onemismatch is present (FIG. 35C) (Hsu et al., 2013). Collectively, theseresults demonstrate that BC-1 can actively target and disrupt the BFPtransgene, and leave negligible off-target footprints. Moreover, whenprecise cuts made by BC-1 are corrected using ssODNs the frequency ofprecise and scarless BFP to GFP edits in Arabidopsis protoplasts isgreater compared to when BC-1 or ssODNs are used alone.

Establishing Precise EPSPS Gene Edits in Flax Using ssODNs andCRISPR/Cas9

To extend the application of genome editing using ssODNs combined withan engineered nuclease to a commercially relevant agricultural crop, aseries of experiments targeting the two highly homologous EPSPS(5′-enolpyruvylshikimate-3-phosphate synthase) loci in flax (Linumusitatissimum) were performed. The EPSPS genes code for a protein in theshikimate pathway that participates in the biosynthesis of aromaticamino acids. In plants, EPSPS is a target for glyphosate, an herbicidethat acts as a competitive inhibitor of the binding site forphosphoenolpyruvate (Schönbrunn et al., 2001). Precise edits in the flaxEPSPS genes were made using ssODNs combined with CRISPR/Cas9 components.A CRISPR/Cas9 expression plasmid (EC-2) that targets a conservedsequence in both EPSPS genes near two loci, T178 and P182, that whenedited to 1178 and A182, will render the EPSPS enzyme tolerant toglyphosate (Gocal et al., 2007). The ssODN EPSPS/144 containing the twotargeted changes, one of which will disrupt the PAM sequence wasintroduced together with EC-2 into flax protoplasts. The treatedprotoplasts were then allowed to divide to form microcolonies withoutusing selection for 21 days (FIG. 7B).

Precise edits and indel scars in both EPSPS loci were identified by PCRamplifying the region surrounding the target site and subjecting theamplicons to deep sequencing. The frequency of precise EPSPS editsranged between 0.09 and 0.23%, and indels between 19.2 and 19.8% inthree independent experiments with these edits and indels being equallydistributed between the two loci:

Summary of flax CRISPR/Cas9 experiments targeting EPSPS Deep sequencingCalli of microcolonies^(a) genotyping results^(c) Experiment PreciseCalli Calli with ID edits (%)^(b) lndels (%) screened precise edits FC-10.23 19.8 5,167 8 (0.15%) FC-2 0.10 19.2 4,601 4 (0.08%) FC-3 0.09 19.6NS ^(a)gDNA was isolated from pools of ~10,000 microcolonies, then usedas template to amplify the target region ^(b)Sequences with T97I (ACA →ATA) and P101A (CCG → GCG); data combined for gene 1 & gene 2^(c)Individual callus was screened first by allele-specific PCR, thenconfirmed by Sanger sequencing NS- Experiment was not screened

After establishing the presence of T178I and P182A edits inmicrocolonies, calli were regenerated, again without employing anyselective agent, then molecularly screened for the targeted edits andindel scars using allele-specific PCR (Morlan et al., 2009). Of 5167calli screened from experiment 1 and 4601 from experiment 2, 8 (0.15%)and 4 (0.08%) contained both T178I and P182A changes in at least one ofthe EPSPS loci respectively. This edit frequency correlated with theinitial sequencing of 21-day-old microcolonies. Calli that screenedpositive for precise edits from this were used to regenerate wholeplants under non-selective conditions-100% of which screened positivefor the presence of the T178I and P182A edits in at least one EPSPS genethrough DNA cloning and Sanger sequencing. All regenerated plantstransferred to soil were fertile and genotyped as heterozygous for theT178I and P182A edits at either the gene 1 or gene 2 locus. No plantswere biallelic or heterozygous for both genes. Cl (conversiongeneration-1) progeny from several A23 line plants derived from a singlecallus event were then evaluated for inheritance of the edited EPSPSallele. Sequence analysis showed sexual transmission of the edited EPSPSallele with the expected Mendelian segregation ratio of 1:2:1:

C₀ Plant Heterozygous for Homozygous for number wt I178 and A182 I178and A182 1 19 31 21 2 15 29 20 3 4 11 2

To identify potential off-target mutations arising from treatment withEC-2 in regenerated plant A23, we amplified 8 different regions of theflax genome bearing sequence similarity to the EC-2 protospacer. NHEJmutations made through imprecise NHEJ events were identified by amplicondeep sequencing. Mutations indicative of EC-2 activity were not detectedin any of the 8 potential off-target sites tested for plant A23:

Off-target analysis of flax plant A23 Off-Target Scaffold or Off-TargetSequencec ^(c) # of Mutations ID Locus ID^(a) Position^(b) (SEQ ID NO)Mismatches detected^(d) Off-1 C7813595 197-219 CcgGTTACAGCAGCaGTCgGCGG 5− (329) Off-2 Lus10030959.g 243476-243460 CcgGTTACAGCAGCaGTCgGCGG 5 −(329) Off-3 Scaffold 155 681644-681624 TcaaaagCtGCAGCTaTCAGTGG 9 − (330)Off-4 Lus10036882.g 1067934-1067911 TcaaaatCtGCAGCTGTCAGTGG 8 − (331)Off-5 Scaffold 107 1077588-1077568 TcaaaatCtGCgGCTGTCAGTGG 9 − (332)Off-6 Scaffold 743 195079-195059 TcaaaatCtGCgGCTGTCAGTGG 9 − (332) Off-7Scaffold 208 238604-238626 AaggacACAGCAGCTGTCgGTGG 7 − (333) Off-8Scaffold 2252 38795-38773 AccaaacgAGCAGCTGTCAGAGG 8 − (334) OnLus10000788.g 19227-19249 GCTGTTACAGCAGCTGTCAGCGG 0 + (335) ^(a)Scaffoldor locus ID from Phytozyme 10.2 ^(b)Protospacer position within scaffold^(c)Lowercase bases are mismatches to the EC-2 protospacer ^(d)Mutationsdetermined by sequencing; On-target mutations are T178I and P182A

Glyphosate Tolerance of Edited Callus and Whole Plants

To determine the glyphosate tolerance afforded by the T178I and P182Amutations, we challenged callus line A23, a line that was identified asbeing heterozygous for the T178I and P182A edits in EPSPS gene 2, aswell as the whole Co plants regenerated from this callus line withglyphosate. A23 callus and control wild type callus was plated on solidregeneration medium containing a range of glyphosate concentrations.After 21 days, the fresh weight of calli with T178I and P182A edits wassignificantly higher (p<0.01) than that of wild type calli at allglyphosate concentrations tested (FIGS. 9A and B). For regenerated wholeplants, both wild type and EPSPS edited plants were maintained in soilunder greenhouse conditions, then sprayed with either 10.5 or 21.0 mMglyphosate. Six days post treatment, wild type plants exhibited a wiltedand necrotic phenotype typical of glyphosate toxicity for bothapplication rates, whereas A23 plants with the edited EPSPS geneexhibited minimal phenotypic change (FIG. 9C). This result is notable asit implies that a single T178I and P182A edited EPSPS gene provides alevel of tolerance much greater than that observed in the controlplants.

Taken together, these data demonstrate that in flax, ssODNs combinedwith CRISPR/Cas9 can result in precise EPSPS edits at sufficientfrequency to be detected by molecular screening without the need forselective culture conditions and that these edits are properlytransmitted to subsequent generations.

RTDS Technology Combined with TALENs to Edit the EPSPS Genes in Flax

To extend the application of RTDS with engineered nuclease mediatedprecision gene editing to other plant systems, a similar study wasperformed targeting the two EPSPS (5′-enolpyruvylshikimate-3-phosphatesynthase) loci in flax. The EPSPS loci encode an enzyme in the shikimatepathway that contributes to the biosynthesis of the aromatic amino acidsphenylalanine, tyrosine and tryptophan. In plants, EPSPS is a target forthe herbicide glyphosate, where it acts as a competitive inhibitor ofthe binding site for phosphoenolpyruvate (Schönbrunn et al., 2001).Based on mutational studies on an E. coli EPSPS homolog, it is expectedthat editing the amino acid positions T97 and P101 to 197 and A101 ofthe flax EPSPS loci will render this enzyme tolerant to glyphosate(Gocal et al., 2007).

In an effort to improve GRON-mediated targeting efficiency for theseEPSPS loci, TALEN LuET-1 (FIG. 38 a that targets conserved sequence forboth EPSPS genes near the T97 and P101 loci (FIG. 38 b ) was designedusing the same architectural guidelines used for BT-1. Transientexpression of LuET-1 protein in flax protoplasts was established using aWestern blot. TALEN protein was detectable 24 h after introduction andremained at a similar level through 48 h (FIG. 38 c ). To apply RTDStechnology in flax, a combination of the TALEN LuET-1 and a 144 nb GRON(EPSPS/144) containing the targeted changes C>T and C>G (ACA>ATA T97I;CCG>GCG P101A) was used. Following these transfections into flaxprotoplasts, precise gene edits as well NHEJ-induced mutations, 7-daypost transfection were analyzed by deep sequencing. Repair events usingNHEJ totaled 1.41%, with deletions and substitutions being most commonand significantly outnumbering insertions (FIG. 39 a ). The majority ofdeletions in the flax EPSPS genes were ≤20 bp (FIG. 39 b ). Using SNPdifferences between the two EPSPS loci (FIG. 40 ), NHEJ events for eachof the two EPSPS loci were found to be comparable, suggesting thatLuET-1 TALEN is effective at cleaving both genes. When examined forprecise, scarless gene edits, 0.19% contained the C>T and C>G targeteddouble change as shown in the following table.

Percentage of total reads Treatment EPSPS gene 1 edits* EPSPS gene 2edits* TALEN + GROW^(†) 0.1 0.09 TALEN alone 0 0 GRON alone 0.02 0.02Mock 0 0 gDNA from treated cells was analyzed by NGS 7 days afterintroduction ^(†)EPSPS/144 GRON *represeats both T97I and P101Amutations in the same read

Similar to the NHEJ repair events, these precise edits had comparablefrequencies for each EPSPS locus indicating that the GRON dependentrepair events are unbiased. These results correlate well with ourArabidopsis BFP to GFP editing data and demonstrate that thecombinatorial approach of GRONs with TALENs in flax protoplastssignificantly increases the frequency of scareless editing of the EPSPSgene targets. Multiple nucleotide edits can be realized with a singleGRON.

Many studies in a variety of different genera, including human, animal,yeast, plant have demonstrated the effectiveness of oligo-directed generepair (Alexeev and Yoon, 1998; Beetham et al., 1999; Kren at al.,(1998); Kuwayama et al., 2008; Li et al., 2001; Rando et al., 1999; Riceet al., 2001; Xiang et al., 1997); Zhu et al., 1999), suggesting that alarge number of genes in a wide variety of organisms are amenable toRTDS.

RTDS-based gene editing in plants can be enhanced when combined with avariety of reagents that create DNA double strand breaks. These datashow in two distinct plant systems, A. thaliana and L. usitatissimum,that by combining GRONs with phleomycin or TALENs the frequency of geneediting is increased markedly when compared to GRONs alone. Thisenhancement can be further increased by altering a GRON's lengthallowing for the added flexibility of targeting several loci with asingle GRON while at the same time increasing the frequency of totaledits. Using an approach that combines TALENs and longer GRONs allowedus to obtain robust precision gene editing frequencies in the flax EPSPSloci to develop glyphosate tolerant traits.

Using RTDS, new non-transgenic breeding traits can be developed inplants with only very minor changes to the target genes and theirresulting proteins. Results presented above show nucleotidesubstitutions in both the BFP transgene in Arabidopsis and the EPSPSgenes in flax. Additionally, this gene editing technology can be appliedrapidly and precisely to improve traits in all commercially relevantcrop plants.

Example 25 Cas9 Protein Delivery in Arabidopsis thaliana

This study investigates the effect of delivering Cas9 protein complexedto gRNA (BFP1) along with GRON to mediate BFP to GFP gene editing inprotoplasts derived from a BFP transgenic Arabidopsis thaliana line. TheGRONs used with the Cas9 RNP contain the coding sequence of the bfp genearound the site of conversion and are labeled with a 2′-O-Me group atthe first 5′ base of the GRON which is a RNA base instead of DNA base.This GRON is herein referred to as 2OMe GRON. Please see Table 1 for adescription of GRON used in these experiments.

BFP transgenic Arabidopsis thaliana protoplasts derived from inducedroot tissue were seeded on a flat-bottom 96-well plate, at 250,000 cellsper well and at a cell density of 1×10⁷ cells/ml. CRISPR-Cas9 wasdelivered as an RNP complex along with GRON by PEG mediated delivery orby alternative delivery methods such as by electroporation,cell-penetrating peptides and/or lipid based delivery techniques.Protoplasts were incubated in the dark at 23° C. for 72 hours, and thenanalyzed by flow cytometry in order to determine the percentage of GFPpositive protoplasts within a given treatment.

The CRISPR-Cas9 consists of two components: Recombinant Streptococcuspyogenes Cas9 (SpCas9) protein and in vitro transcribed sgRNA. The sgRNAis a fusion of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA).The crRNA region contains the spacer sequence used to guide the Cas9nuclease to the BFP target gene. In these experiments the BFP1CRISPR-Cas9 which targets the bfp gene was used. The GRON contains thecoding sequence of the bfp gene near the site of conversion. Table 1describes the GRON and Table 2 describes the BFP gRNA used in theseexperiments.

Results

Delivery of Cas9 protein complexed with gRNA (BFP1), when used incombination with GRON (2OMe; BFP4/NC 101-mer), resulted in 2.20-3.10%BFP to GFP gene editing from three independent experiments. Controltreatments without GRON resulted in no detectable BFP to GFP geneediting. These data demonstrate the advantages in using the GRON forprecision gene editing.

Example 26 Cas9 Protein Delivery in Brassica napus

This study investigates the effect of delivering Cas9 protein complexedto gRNA (BnEPSPS gRNA-1) along with GRON to mediate EPSPS gene editingin protoplasts derived from Brassica napus leaf material. The GRONs usedwith the Cas9 RNP contains the coding sequence of the targeted epspsgene around the site of conversion and are labeled with a 2′-O-Me groupat the first 5′ base of the GRON which is a RNA base instead of DNAbase. These GRONs are herein referred to as 2OMe GRONs.

Brassica napus protoplasts derived from leaves of in vitro propagatednodal cuttings were seeded in a 50 ml centrifuge tube at 2,000,000 cellsper tube and at a cell density of 5×10⁶ cells/ml. CRISPR-Cas9 targetingthe EPSPS 2-25 gene along with GRON was delivered as a ribonucleoproteincomplex using the PEG method or by alternative delivery methods such asby electroporation, cell-penetrating peptides and/or lipid baseddelivery techniques. Protoplasts were incubated in the dark at 25° C.for three weeks, and then analyzed deep sequencing in order to determinethe percentage of precise EPSPS edits and frequency of indels within agiven treatment.

The CRISPR-Cas9 consists of two components: the Streptococcus pyogenesCas9 (SpCas9) and sgRNA which form the RNP CRISPR-Cas9 complex whenmixed. The sgRNA is a fusion of CRISPR RNA (crRNA) and trans-activatingcrRNA (tracrRNA). The crRNA region contains the spacer sequence used toguide the Cas9 nuclease to the EPSPS target gene. In these experimentsRNP CRISPR-Cas9 is used to target the EPSPS 2-25 gene. The GRON containsthe coding sequence of the EPSPS gene near the site of conversion aswell as nucleotide alterations to produce the desired changes to theEPSPS gene.

BnEPSPS-2-25 101-mer 2OMe (SEQ ID NO: 336)5′(U)TACCTTGCGTTGCCACCTGCAGCAGTAACTGCAGCGGTAAGTGTACGCATCGCTATTCCAGCATTCCC AAGGTACAACTCGATATCACTCTTGGAATCTA3′BnEPSPS gRNA-1 (SEQ ID NO: 337) 5′GCAGCGGTAAGTGGACGCA 3′

Results

Delivery of Cas9 protein complexed with gRNA (BnEPSPS gRNA-1) when usedin combination with GRON (2′OMe; BnEPSPS-2-25/NC 101-mer) resulted in0.07-0.125% gene editing and 27.1-39.4% indel formation in the targetedepsps gene(s) from three independent experiments. Control treatmentswithout GRON resulted in no detectable EPSPS gene editing demonstratingthe advantages in using the GRON for precision gene editing.

Example 27 Cas9 Protein Delivery in Oryza sativa

This study investigates the effect of delivering Cas9 protein complexedto gRNA (CR-OsACCase-4) to mediate targeted indel formation around theCas9 DNA cleavage site in the accase gene in protoplasts derived fromrice cell suspensions. Please see Table 1 for a description of gRNAsused in these experiments.

Oryza sativa protoplasts derived from cell suspensions were treated in a0.4 cm cuvette at 1×10⁶ cells per cuvette and at a cell density of 1×10⁶cells/ml. Ribonucleoprotein complexes consisting of Cas9 protein and invitro synthesized gRNA along with GRON was introduced into protoplastsby electroporation. Protoplasts were incubated in the dark at 23° C. for72 h, and then analyzed by T7E1 assay in order to determine thepercentage of indels derived from the activity of the CRISPR-Cas9protein.

CR-OsACCase-4 (SEQ ID NO: 338) 5′-AGAGCTACGAGGAGGGGCTT-3′

Results

Delivery of Cas9 protein complexed with gRNA (CR-OsACCase-4) resulted in15.9-23.3% indel formation respectively around the site of Cas9 cleavageof the accase gene from three independent experiments. These datademonstrate the functionality of Cas9 for targeted DNA activity whendelivered into protoplasts as a protein.

Example 28 Cas9 Protein Delivery in Cassava

This study investigates the effect of delivering Cas9 protein complexedto gRNA (BFP1) along with GRON to mediate BFP to GFP gene editing inprotoplasts derived from cassava cell suspension cultures. The GRONsused with the Cas9 RNP contain the coding sequence of the targeted bfpgene around the site of conversion and are labeled with a 2′-O-Me groupat the first 5′ base of the GRON which is a RNA base instead of DNAbase. These GRONs are herein referred to as 2′OMe GRONs. Please seeTable 1 for a description of GRONs used in these experiments.

Cassava protoplasts derived from BFP transgenic FEC (friable embryogeniccallus) suspension cultures were seeded in 14 ml centrifuge tubes, at4×10⁶ cells per tube at a cell density of 5×10⁶ cells/ml. TheCRISPR-Cas9 RNP was added at 25 μg/million cells. The GRON (along withthe CRISPR-Cas9 RNP) was introduced into protoplasts by PEG mediateddelivery of 0.5 μM of the BFP4/NC 201-mer GRON. Protoplasts wereincubated in the dark at 25° C. for 72 hours, and then they wereanalyzed by flow cytometry in order to determine the percentage of GFPpositive protoplasts within a given treatment.

Results

Delivery of Cas9 protein complexed with gRNA (BFP1), when used incombination with GRON (2′OMe; BFP4/NC 201-mer), resulted in 0.008-0.009%BFP to GFP gene editing. Control treatments without GRON resulted in nodetectable BFP to GFP gene editing. These data demonstrate theadvantages in using the GRON for precision gene editing.

Example 29 Cas9 Protein Delivery in Solanum tuberosum

This study investigates the effect of delivering Cas9 protein complexedto gRNA (PPX2 or PPX4) to mediate targeted indel formation around theCas9 DNA cleavage site in the ppx gene in protoplasts derived frompotato leaf material. Please see Table 1 for a description of gRNAs usedin these experiments.

Protoplasts of the Solanum tuberosum ST-01 plants were mixed with Cas9protein complexed with guide RNAs designed for the 144 (PPX2) or the 220(PPX4) position of the potato protoporphyrinogen gene. Protoplasts wereincubated with the Cas9/gRNA complex for 15 minutes on ice prior to thetreatment with the transfection reagent, Polyethylene glycol. Uponmixing all components, the tubes were incubated on ice for 30 minutesprior to dilution with a salt solution osmotically adjusted to maintainthe stability of the protoplasts. The protoplasts were centrifuged at44.16×g for 10 minutes and suspended in 1 ml of culture medium and wereincubated at 22° C. At 24 hours post transfection, genomic DNA wasisolated from the samples and submitted for NGS (Next GenerationSequencing) analysis.

PPX2 (SEQ ID NO: 280) 5′-GCCTTCCACAAGACAAAGCG-3′ PPX4 (SEQ ID NOS: 281)5′-GCTCAATTTTGAGGGGTCAC-3′

Results

Delivery of Cas9 protein complexed with gRNA (PPX2 or PPX4) resulted inup to 24.4 and 35.7% indel formation respectively around the site ofCas9 cleavage of the ppx gene. These data demonstrate the functionalityof Cas9 for targeted DNA activity when it is delivered into protoplastsas a protein.

Example 30 Cas9 mRNA Delivery in Arabidopsis thaliana

This study investigates the effect of delivering Cas9 mRNA and gRNA(BFP1) along with GRON to mediate BFP to GFP gene editing in protoplastsderived from a BFP transgenic Arabidopsis thaliana line. The GRONs usedwith the Cas9 RNP contain the coding sequence of the bfp gene around thesite of conversion and are labeled with a 2′-O-Me group at the first 5′base of the GRON which is an RNA base instead of DNA base. This GRON isherein referred to as 2OMe GRON.

The CRISPR-Cas9 consisted of two components: Streptococcus pyogenes Cas9(SpCas9) mRNA and in vitro transcribed sgRNA. The sgRNA is a fusion ofCRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The crRNAregion contains the spacer sequence used to guide the Cas9 nuclease tothe BFP target gene. In these experiments the BFP1 CRISPR-Cas9 is usedwhich targets the bfp gene. The GRON contains the coding sequence of thebfp gene near the site of conversion.

BFP transgenic Arabidopsis thaliana protoplasts derived from inducedroot tissue were seeded on a flat-bottom 96-well plate, at 250,000 cellsper well and at a cell density of 1×10 cells/ml. CRISPR-Cas9 wasdelivered as RNA species (Cas9 mRNA and sgRNA) along with GRON by PEGmediated delivery or by alternative delivery methods such as byelectroporation, cell-penetrating peptides and/or lipid based deliverytechniques. Protoplasts were incubated in the dark at 23° C. for 72hours, and then analyzed by flow cytometry in order to determine thepercentage of GFP positive protoplasts within a given treatment.

Results

Delivery of Cas9 mRNA and gRNA (BFP1) when used in combination with GRON(2OMe; BFP4/NC 101-mer) resulted in 0.24-1.51% BFP to GFP gene editingfrom three independent experiments. Control treatments without GRONresulted in no detectable BFP to GFP gene editing. These datademonstrate the advantages in using the GRON for precision gene editing.

REFERENCES

-   Alexeev, V., and Yoon, K. (1998) Stable and inheritable changes in    genotype and phenotype of albino melanocytes in deuced by an RNA-DNA    oligonucleotide. Nat. Biotechnology, 16, 1343-1346.-   Ansai, S., Sakuma, T., Yamamoto, T., Ariga, H., Uemura, N.,    Takahashi, R. and Kinoshita, M. (2013) Efficient targeted    mutagenesis in medaka using custom-designed transcription    activator-like effector nucleases. Genetics, 193, 739-749.-   Aarts, M., Dekker, M., de Vries, S., van der Wal, A., and te    Riele, H. (2006) Generation of a mouse mutant by    oligonucleotide-mediated gene modification in ES cells. Nucleic    Acids Res., 34, e147-e147.-   Aryan, A., Anderson, M. A. E., Myles, K. M. and    Adelman, Z. N. (2013) TALEN-based gene disruption in the dengue    vector Aedes aegypti. PLoS ONE, 8 (3), e60082.-   Bedell, V. M., Wang, Y., Campbell, J. M., Poshusta, T. L.,    Starker, C. G., Krug, R. G 2^(nd), Tan, W., Penheiter, S. G., Ma, A.    C., Leung, A. Y., Fahrenkrug, S. C., Carlson, D. F., Voytas, D. F.,    Clark, K. J., Essner, J. J. and Ekker, S. C. (2012) In vivo genome    editing using a high-efficiency TALEN system. Nature, 491, 114-118.-   Beetham, P. R., Kipp, P. B., Sawycky, X. L., Arntzen, C. J., and    May, G. D. (1999) A tool for functional plant genomics: chimeric    RNA/DNA oligonucleotides cause in vivo gene-specific mutations.    Proc. Natl. Acad. Sci. USA, 96, 8774-8778.-   Binding, H and Nehls, R. (1977) Regeneration of isolated protoplasts    to plants in Solanum dulcamara L. Z. Pflanzenphysiol, 85, 279-280.-   Blum, R. H., Carter, S. K., and Agre, K. (1973) A clinical review of    bleomycin-a new antineoplastic agent. Cancer, 31, 903-914.-   Carlson, D. F., Tan, W., Lillico, S. G., Stverakova, D., Proudfoot,    C., Christian, M., Voytas, D. F., Long, C. R., Whitelaw, C. B., and    Fahrenkrug, S. C. (2012) Efficient TALEN-mediated gene knockout in    livestock. Proc. Natl. Acad. Sci. USA, 109, 17382-17387.-   Cermak, T., Doyle, E. L., Christian, M., Wang, L., Zhang, Y.,    Schmidt, C., Baller, J. A., Somia, N. V., Bogdanove, A. J., and    Voytas, D. F. (2011) Efficient design and assembly of custom TALEN    and other TAL effector-based constructs for DNA targeting. Nucleic    Acids Res., 39, e82.-   Gibson, D. G., Young, L., Chuang, R. Y., Venter, J. C., Hutchison    3^(rd), C. A. and Smith, H. O. (2009) Enzymatic assembly of DNA    molecules up to several hundred kilobases Nat. Methods, 6 343-345.-   Gamborg, O. L., Miller, R. A., and Ohyama, K. (1968) Nutrient    requirements of suspension cultures of soybean root cells. Exp Cell    Res., 50, 151-8.-   Giloni, L., Takeshita, M., Johnson, F., Iden, C., and    Grollman, A. P. (1981) Bleomycin-induced strand-scission of DNA:    mechanism of deoxyribose cleavage. J. Biol. Chem., 256 8608-8615.-   Gocal, F. W., Schöpke, C., Beetham, P. R. (2015) Oligo-mediated    targeted gene editing. In Zhang, F., Puchta, H., and Thomson, J. G.    (Eds), Advances in New Technology for Targeted Modification of Plant    Genomes. Springer. In print-   Gocal, G., Knuth, M., and Beetham, P. (2007) Generic EPSPS mutants.    U.S. Pat. No. 8,268,622. Filled Jan. 10, 2007, Issued Sep. 18, 2012.-   Kren, B. T., Bandypadhyay, P., and Steer, C. J. (1998) In vivo    site-directed mutagenesis of the factor IX gene by chimeric RNA/DNA    oligonucleotides. Nat. Medicine, 4, 285-290.-   Kuwayama H., Yanagida T., and Ueda, M. (2008) DNA    oligonucleotide-assisted genetic manipulation increases    transformation and homologous recombination efficiencies: evidence    from gene targeting of Dictyostelium discoideum. J. Biotechnol.,    133, 418-423.-   Lei, Y., Guo, X., Deng, Y., Chen, Y., Zhao, H. (2013) Generation of    gene disruptions by transcription activator-like effector nucleases    (TALENs) in Xenopus tropicalis embryos. Cell Biosci., 3, 21.-   Liang, Z., Zhang, K., Chen, K., and Gao, C. (2013) Targeted    Mutagenesis in Zea mays using TALENs and the CRISPR/Cas System. J    Genet Genomics, 41, 63-68.-   Li, T., Huang, S., Zhao, X., Wright, D. A., Carpenter, S.,    Spalding, M. H., Weeks, D. P., and Yang, B. (2011) Modularly    assembled designer TAL effector nucleases for targeted gene knockout    and gene replacement in eukaryotes. Nucleic Acids Res., 39,    6315-6325.-   Li, T., Liu, B., Spalding, M. H., Weeks, D. P., and Yang, B. (2012)    High-efficiency TALEN-based gene editing produces disease-resistant    rice. Nat. Biotechnol., 30, 390 e392.-   Li, Z. H., Liu, D. P., Yin, W. X., Guo, Z. C., and    Liang, C. C. (2001) Targeted correction of the point mutations of    b-thalassemia and targeted mutagenesis of the nucleotide associated    with HPFH by RNA/DNA oligonucleotides: potential for b-thalassemia    gene therapy. Blood Cells Mol. Dis., 27, 530-538.-   Lieber, M. R. (2010) The mechanism of double-strand DNA break repair    by the nonhomologous DNA end-joining pathway. Annu. Rev. Biochem.,    79, 181-183.-   Liu, J., Li, C., Yu, Z., Huang, P., Wu, H., Wei, C., Zhu, N., Shen,    Y., Chen, Y., Zhang, B., Deng, W. M. and Jiao, R. (2012) Efficient    and specific modifications of the Drosophila genome by means of an    easy TALEN strategy. J. Genet. Genomics 39, 209-215.-   Lor, V. S., Starker, C. G., Voytas, D. F., Weiss. D., and    Olszewski, N. E. (2014) Targeted mutagenesis of the tomato PROCERA    gene using transcription activator-like effector nucleases. Plant    Physiol., 166, 1288-91.-   Mathur, J. and Koncz, C. A (1995) Simple method for isolation,    Liquid culture, transformation and regeneration of Arabidopsis    thaliana protoplasts. Plant Cell Rep., 10, 221-226.-   Menczel L., Nagy F., Kiss Z. R., and Maliga P. (1981). Streptomycin    resistant and sensitive somatic hybrids of Nicotiana    tabacum+Nicotiana knightiana-correlation of resistance to N. tabacum    plastids. Theor. Appl. Genet., 59, 191-195.-   Menke, M., Chen, I. P., Angelis, K. J., and Schubert I. (2001) DNA    damage and repair in Arabidopsis thaliana as measured by the comet    assay after treatment with different classes of genotoxins. Mutat.    Res., 493, 87-93.-   Moerschell, R. P., Tsunasawa, S., and Sherman F. (1988)    Transformation of yeast with synthetic oligonucleotides. Proc. Natl.    Acad. Sci. USA, 85, 524-528.-   Murashige, T. and Skoog, F. A. (1962) A revised medium for rapid    growth and bio-assays with tobacco tissue cultures. Physiol Plant,    15, 473-497.-   Mussolino, C., Alzubi, J., Fine, E. J., Morbitzer, R., Cradick, T.    J., Lahaye, T., Bao, G. and Cathomen, T. (2014) TALENs facilitate    targeted genome editing in human cells with high specificity and low    cytotoxicity. Nucleic Acids Res., 42, 6762-6773.-   Qiu, Z., Liu, M., Chen, Z., Shao, Y., Pan, H., (2013)    High-efficiency and heritable gene targeting in mouse by    transcription activator-like effector nucleases. Nucleic Acids Res.,    41, e120.-   Rando, T. A., Disatnik, M. H. and Zhou, L. Z. (2000) Rescue of    dystrophin expression in mdx mouse muscle by RNA/DNA    oligonucleotides. Proc. Natl Acad. Sci., USA, 97, 5363-5368.-   Rice, M. C., Bruner, M., Czymmek, C., and Kmiec, E. B. (2001) In    vitro and in vivo nucleotide exchange directed by chimeric RNA/DNA    oligonucleotides in Saccharomyces cerevisae. Mol. Microbiology, 40,    857-868.-   Rivera-Torres, N., Strouse, B., Bialk, P., Niamat, R. A.,    Kmiec, E. B. (2014) The position of DNA cleavage by TALENs and cell    synchronization influences the frequency of gene editing directed by    single-stranded oligonucleotides. PLoS ONE, 9, e96483-   Roger, D., David., A., and David, H. (1996) Immobilization of flax    protoplasts in agarose and alginate Beads. Plant Physiol., 112,    1191-1199.-   Schönbrunn, E., Eschenburg, S., Shuttleworth, W. A., Schloss, J. V.,    Amrhein, N., and Evans, J. N. S. (2001) Interaction of the herbicide    glyphosate with its target enzyme 5-enolpyruvylshikimate 3-phosphate    synthase in atomic detail. Proc Natl. Acad. Sci., 98, 1376-80.-   Schröpfer, S., Knoll, A., Trapp, O., and Puchta, H. (2014) DNA    repair and recombination in plants. Springer New York: The Plant    Sciences, Volume 2, 51-93.-   Shan, Q., Wang, Y., Chen, K., Liang, Z., Li, J., Zhang, Y., Zhang,    K., Liu, J., Voytas, D. F., Zheng, X., Zhang, Y., and Gao,    C., (2013) Rapid and efficient gene modification in rice and    Brachypodium using TALENs. Mol. Plant, 6, 1365-1368.-   Shan, Q., Zhang, Y., Chen, K., Zhang, K., and Gao, C. (2015)    Creation of fragrant rice by targeted knockout of the OsBADH2 gene    using TALEN technology. Plant Biotechnol J., doi: 10.1111/pbi.12312.-   Sung, Y. H., Baek, I.-J., Kim, D. H., Jeon, J., and Lee, J. (2013)    Knockout mice created by TALEN-mediated gene targeting. Nat.    Biotechnol., 31, 23-24.-   Strouse, B., Bialk, P., Niamat, R., Rivera-Torres, N., and    Kmiec, E. B. (2014) Combinatorial gene editing in mammalian cells    using ssODNs and TALENs. Scientific Reports, 4, 3791.-   Suzuki, T., Murai, A., and Muramatsu, T. (2003) Low-dose bleomycin    induces targeted gene repair frequency in cultured melan-c cells    using chimeric RNA/DNA oligonucleotide transfection. Int. J. Mol.    Med., 12, 109-114.-   Symington, L. S. and Gautier, J. (2011) Double-strand break end    resection and repair pathway choice. Annu. Rev. Genet., 45, 247-71.-   Voytas, D. F. (2013) Plant genome engineering with sequence-specific    nucleases. Annu. Rev. Plant Biol., 64, 327-50.-   Wendt, T., Holm, P. B., Starker, C. G., Christian, M., Voytas, D.    F., Brinch-Pedersen, H., and Holme, I. B. (2013) TAL effector    nucleases induce mutations at a pre-selected location in the genome    of primary barley transformants. Plant Mol. Biol., 83, 279-285.-   Xiang, Y., Cole-Strauss, A., Yoon, K., Gryn, J., and Kmiec, E.    B, (1997) Targeted gene conversion in a mammalian CD34+-enriched    cell population using a chimeric RNA/DNA oligonucleotide. J. Mol.    Med., 75, 829-835.-   Yoon, K., Cole-Strauss, A., and Kmiec, E. B. (1996) Targeted gene    correction of episomal DNA in mammalian cells mediated by a chimeric    RNA. DNA oligonucleotide. Proc. Natl. Acad. Sci. USA, 93, 2071-2076.-   Zhang, H., Gou, F., Zhang. J., Liu, W., Li, Q., Mao, Y., Botella, J.    R., and Zhu, J. K. (2015) TALEN-mediated targeted mutagenesis    produces a large variety of heritable mutations in rice. Plant    Biotechnol J. April 13. doi: 10.1111/pbi.12372.-   Zhang, Y., Zhang, F., Li, X., Baller, J. A., Qi, Y., Starker, C. G.,    Bogdanove, A. J., and Voytas, D. F. (2013) Transcription    activator-like effector nucleases enable efficient plant genome    engineering. Plant Physiol., 161, 20-27.-   Zhu T., Peterson D. J., Tagliani L., St. Clair G., Baszczynski C.    L., and Bowen B. (1999) Targeted manipulation of maize genes in vivo    using chimeric RNA/DNA oligonucleotides. Proc. Natl. Acad. Sci. USA,    96, 8768-8773.

One skilled in the art readily appreciates that the present disclosureis well adapted to carry out the objects and obtain the ends andadvantages mentioned, as well as those inherent therein. The examplesprovided herein are representative of preferred embodiments, areexemplary, and are not intended as limitations on the scope of thedisclosure.

It will be readily apparent to a person skilled in the art that varyingsubstitutions and modifications may be made to the disclosure disclosedherein without departing from the scope and spirit of the disclosure.

The disclosure illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the disclosure claimed. Thus, it should be understood thatalthough the present disclosure has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this disclosure as defined by the appended claims.

Thus, it should be understood that although the present disclosure hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification, improvement, and variation of the disclosuresdisclosed may be resorted to by those skilled in the art, and that suchmodifications, improvements and variations are considered to be withinthe scope of this disclosure. The materials, methods, and examplesprovided here are representative of preferred embodiments, areexemplary, and are not intended as limitations on the scope of thedisclosure.

The disclosure has been described broadly and generically herein. Eachof the narrower species and subgeneric groupings falling within thegeneric disclosure also form part of the disclosure. This includes thegeneric description of the disclosure with a proviso or negativelimitation removing any subject matter from the genus, regardless ofwhether or not the excised material is specifically recited herein.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

All publications, patent applications, patents, and other referencesmentioned herein are expressly incorporated by reference in theirentirety, to the same extent as if each were incorporated by referenceindividually. In case of conflict, the present specification, includingdefinitions, will control.

Other embodiments are set forth within the following claims.

What is claimed is:
 1. A method of causing one or more targetedmutations in a flax cell, said method comprising: delivering a generepair oligonucleobase (GRON) into the flax cell, wherein the GRON is anucleic acid sequence that hybridizes at a target DNA sequence within anEPSPS gene in the flax cell genome and comprises one or more mismatchedbase-pair(s) relative to the target DNA sequence that encodes thetargeted mutation(s), wherein the targeted mutation(s) are selected toincrease glyphosate resistance in flax; and culturing the flax cellunder conditions that increase one or more cellular DNA repair processesprior to, coincident with, or both prior to and coincident with deliveryof a GRON into the plant cell, wherein the conditions that increase oneor more cellular DNA repair processes comprise introducing one or moreTALENs or CRISPR nucleases into the flax cell; identifying one or moreflax cells comprising the targeted mutations(s) without herbicideselection by next generation sequencing of the EPSPS gene in the one ormore flax cells, wherein the one or more flax cells are non-transgenicwith regard to the targeted mutations(s); and regenerating a flax plantfrom the one or more identified flax cells, wherein the flax plant isnon-transgenic with regard to the targeted mutations(s) and exhibitsglyphosate resistance.
 2. The method of claim 1, wherein the GRONcomprises DNA and RNA nucleotides.
 3. The method of claim 1, whereinsaid GRON is single stranded.
 4. The method of claim 3, wherein the GRONis chemically protected at the 5′ end, at the 3′ end, or at the 5′ and3′ ends.
 5. The method of claim 4, wherein the chemical protectioncomprises one or more moieties selected from an idC group, a Cy3 group,a group comprising three phosphorothioate internucleotide linkages, anda 2′-O-methyl group.
 6. The method of claim 4, wherein the chemicalprotection comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 RNA bases at the5′ end.
 7. The method of claim 1, wherein said GRON is single strandedand has a wobble base relative to the target sequence for the geneticchange.
 8. The method of claim 1, wherein said GRON is between 50 and201 nucleotides in length.
 9. The method of claim 1 wherein theconditions that increase one or more cellular DNA repair processescomprise a CRISPR nuclease.
 10. The method of claim 1, wherein thetargeted mutation(s) comprise a threonine to isoleucine substitutioncorresponding to T1781 and a proline to alanine substitutioncorresponding to P182A, with amino acid numbering relative to the aminoacid sequence of the Flax EPSPS protein sequence of SEQ ID NO:
 12. 11.The method of claim 10, wherein the plant comprises an EPSPS gene thatis homozygous for the T1781 and P182A substitutions.